Autoantigenic fragments, methods and assays

ABSTRACT

The present invention provides a method of producing autoantigens, compositions comprising autoantigenic fragments and methods of using autoantigenic fragments in the treatment of a condition associated with an autoimmune response. Also provided are assays for the detection or assessment of an autoimmune response.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 09/296,662, filed Apr. 22, 1999, now allowed.

STATEMENT REGARDING FEDERALLY-SPONSORED R&D

This invention was made in part under Federally Sponsored Research. TheU.S. Government may have certain rights in this invention.

REFERENCE TO MICROFICHE APPENDIX

Not applicable.

FIELD OF THE INVENTION

This invention relates to the production of autoantigenic fragments fromautoantigens and the uses of the autoantigenic fragments.

BACKGROUND OF THE INVENTION

The mature immune system of animals differentiates betweenself-molecules and non-self-molecules and mounts an immune response onlyagainst the latter. The immune system learns which molecules are selfthrough constant exposure to those molecules that are normally a part ofthe animal. Thus, the mature immune system is tolerized to the presenceof molecules that are self. However, the immune system is not tolerizedto molecules that are newly presented in the animal. These molecules canbe antigens and thereby stimulate an immune response against them.Commonly, newly presented antigens are from an extracorporeal source,such as an infection. In this case, the immune response helps to destroythe source of the antigens and thereby clear the infection from thebody.

Newly presented antigens are produced in vivo through the degradation ofcellular components. When the immune system recognizes these degradationproducts of self molecules as “non-self” antigens, an immune responsecan be mounted against them and an autoimmune disease can develop. Thus,these antigens are members of the class of molecules generally referredto as autoantigens and the antibodies produced against them are referredto as autoantibodies. For clarity herein, autoantigen is used to referto the complete self molecule as found in the body. Autoantigenicfragment is used to refer to the degradation product of the autoantigen.Thus it is when an epitope is presented to the immune system asautoantigenic fragment that an immune response is elicited. Onceelicited, the immune response can target the autoantigenic fragment, theautoantigen, or both.

Autoimmune diseases are diseases in which a specific immune response toself-molecules occurs, often leading to tissue and organ damage anddysfunction. The diseases can be organ-specific (e.g. Type I diabetesmellitus, thyroiditis, myasthenia gravis, primary biliary cirrhosis) orsystemic in nature (e.g. systemic lupus erythematosus, rheumatoidarthritis, polymyositis, dermatomyositis, Sjogrenís syndrome,scleroderma, and graft-vs.-host disease).

One source of autoantigenic fragments is cleavage of an autoantigenduring apoptosis. Apoptosis is a morphologically and biochemicallydistinct form of cell death that occurs in many different cell typesduring a wide range of physiologic and pathologic circumstances(reviewed in (Jacobson et al., 1997; Thompson, 1995; White, 1996)).Studies report that specific proteolysis catalyzed by a novel family ofcysteine proteases is of critical importance in mediating apoptosis(Chinnaiyan and Dixit, 1996a; Martin and Green, 1995; thornberry andMolineaux, 1995). These proteases (termed caspases), cleave downstreamsubstrates after a consensus tetrapeptide sequence ending with asparticacid (P₁). The caspases are synthesized as inactive precursors thatrequire specific proteolytic cleavage after an aspartic acid residue foractivation (reviewed in (Nicholson and Thornberry, 1997)).

Granzyme B, a serine protease found in the cytoplasmic granules ofcytotoxic T lymphocytes (CTL) and natural killer (NK) cells, has asimilar requirement to caspases, for aspartic acid in the substrate P₁position (Odake et al., 1991; Poe et al., 1991). Studies have reportedthat granzyme B plays an important role in inducing apoptotic nuclearchanges in target cells during granule exocytosis induced cytotoxicity(Darmon et al., 1996; Heusel et al., 1994; Sarin et al., 1997; Shrestaet al., 1995; Talanian et al., 1997).

Granzyme B is described as catalyzing the cleavage and activation ofseveral caspases (Chinnaiyan et al., 1996b; Darmon et al., 1995; Duan etal., 1996; Fernandes-Alnemri et al., 1996; Gu et al., 1996; Martin etal., 1996; Muzio et al., 1996; Quan et al., 1996; Sarin et al., 1997;Song et al., 1996a; Srinivasula et al., 1996; Talanian et al., 1997;Wang et al., 1996). Granzyme B also initiates caspase-independentpathways which contribute to target cell death. However, while severalcandidates for these additional pathways exist, they remain largelyundefined (Sarin et al., 1997; Talanian et al., 1997).

One candidate pathway is the direct proteolysis of death substrates bygranzyme B, although efficient non-caspase cellular substrates for thisprotease have not yet been identified. Initial studies have indicatedthat the cleavage of PARP, U1-70 kDa and lamin B observed duringgranzyme B-induced cell death is catalyzed by caspases, rather thandirectly by granzyme B (Darmon et al., 1995; Martin et al., 1996;Talanian et al., 1997), but the effects of granzyme B on other caspasesubstrates in vitro and during granule-induced cytotoxicity have notbeen extensively studied.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Granzyme B cleaves purified DNA-PK_(CS), NuMA, PARP and caspases3 and 7 in vitro with different efficiencies. Reactions containing0-12.5 nM granzyme B (for DNA-PK_(CS) and NuMA)or 0-50 nM granzyme B(for caspases 3 and 7 and PARP) were performed as described inExample 1. Cleavage fragments were detected by fluorography orimmunoblotting (monoclonal antibody 18-2 was used to detectDNA-PK_(CS)). On the right side of each panel, the SDS-PAGE migrationpositions of the intact molecules are denoted by arrows, and thefragment sizes are indicated.

FIG. 2. Cleavage of autoantigens in vitro with purified granzyme B orcaspase-3 yields different fragments. Reactions containing 30 nMpurified DNA-PK_(CS) (lanes 1-3), endogenous DNA-PK_(CS) and NuMA inHeLa lysate (lanes 4-9) or [³⁵S]methionine-labeled PARP (lanes 10-12)were incubated with the following amounts of granzyme B: 1.25 nM (lane2); 12.5 nM (lanes 5 & 8) or 50 nM (lane 11). Similar experiments wereperformed with these substrates using the following amounts ofcaspase-3: 42 pM (lanes 3 &12) or 100 pM (lanes 6 & 9). Reactionmixtures were incubated at 37° C. for 15 min. (granzyme B reactions) or60 min. (caspase-3 reactions). Note that 100 nM Ac-DEVD-CHO was addedwhen granzyme B was used to cleave DNA-PK_(CS) and NuMA in HeLa celllysates, to prevent activation of HeLa cell caspases. Intact and cleavedNuMA and DNA-PK_(CS) were visualized by immunoblotting, and PARP wasvisualized by autoradiography. In the case of DNA-PK_(CS), blotsobtained using monoclonal antibodies 25-4 (directed against theC-terminus) and 18-2 (directed against the N-terminus) are shown inlanes (1-3) and (4-6), respectively.

FIG. 3. Kinase activity of DNA-PK_(CS) is abolished after cleavage withgranzyme B. Kinase assays were performed using intact DNA-PK_(CS) (lanes1 & 2) or granzyme B-cleaved DNA-PK_(CS) (lanes 3 & 4) in the absence(lanes 2 & 4) or presence (lanes 1 & 3) of 10 μg/ml of DNA. DNA-PK_(CS)itself was omitted from the otherwise complete kinase reaction mix as acontrol in lane 5. Phosphorylation of SP1 substrate was detected byautoradiography.

FIG. 4. Endogenous DNA-PK_(CS) and NuMA in HeLa cell lysates are cleavedin a caspase-independent manner after adding purified granzyme B andincubating in vitro. 12.5 nM purified granzyme B (lanes 5-7) or 105 pMpurified caspase-3 (lanes 2-4) were added to lysates of control HeLacells, in the presence of 100 nM Ac-DEVD-CHO (lanes 3 & 6) or 100 nMAc-YVAD-CHO (lanes 4 & 7). After incubating at 37° C. for 60 min, thereactions were terminated. DNA-PK_(CS), NuMA and PARP were detected byimmunoblotting as described in Example 1 (monoclonal antibody 18-2 wasused to detect DNA-PK_(CS)). Equal amounts of protein wereelectrophoresed in each lane.

FIG. 5. Granzyme B cleaves DNA-PK_(CS) at VGPD²⁶⁹⁸-F²⁶⁹⁹ SEQ ID NO: 24and DEVD²⁷¹²-N²⁷¹³ SEQ ID NO: 1. A [³⁵S]methionine-labeled wild-type(wt) DNA-PK_(CS) polypeptide (DNA-PK_(CS) ²⁵⁶⁶-²⁹²⁸) and tworadiolabeled polypeptides containing mutations in the P₁ positions ofthe predicted granzyme B cleavage site (D²⁶⁹⁸A) and the known caspase-3cleavage site (D²⁷¹²A), were generated as described in Example 1. Thesepolypeptides were incubated in the absence of added proteases (lanes 1,4 & 7), or in the presence of 8 nM recombinant caspase-3 (lanes 2, 5 &8) or 8 nM purified granzyme B (lanes 3, 6 & 9) for 60 min at 37° C.After terminating the reactions, samples were electrophoresed, and theintact polypeptide and the cleavage products were detected byfluorography. Fragment sizes of 18 kDa (a), 28 kDa (b), 20 kDa (c) and26 kDa (d) were generated (see lower panel for schematicrepresentation).

FIG. 6. Endogenous DNA-PK_(CS), NuMA and PARP are cleaved after in vivoincubation of intact K562 cells with YT cell granule contents. K562cells were incubated for 30 min at 37° C. in the absence (lanes 1-3) orpresence (lane 4) of 100 μM Ac-DEVD-CHO. Aliquots of these cellsuspensions (each containing 3×10⁵ K562 cells) were then furtherincubated for 90 min at 37° C. in the presence of 1 mM Ca²⁺ (lane 1), 1mM EDTA+YT cell granule contents (lane 2) or 1 mM Ca²⁺+YT cell granulecontents (lanes 3 & 4). After terminating the reactions, DNA-PK_(CS),NuMA and PARP were detected by immunoblotting as described in theExample 1 (Patient serum G.A. was used to blot DNA-PK_(CS)).

FIG. 7. Granzyme B-specific fragment of DNA-PK_(CS) is generated in K562cells attacked by LAK cells. Fas-negative K562 target cells werepreincubated in the absence (lanes 1-3) or presence (lane 4) of 100 μMAc-DEVD-CHO for 1 hr, followed by co-incubation for a further 4 hr at37° C. (effector:target ratio 5:1). After terminating the reactions, thefollowing numbers of cells were electrophoresed in each gel lane:1.7×10⁶ LAK cells (lane 1); 0.34×10⁶ K562 cells (lane 2); 1.7×10⁶ LAKcells plus 0.34×10⁶ K562 cells (lanes 3 & 4). DNA-PK_(CS) and PARP weredetected by immunoblotting; patient serum G.A. was used to detectDNA-PK_(CS).

FIG. 8. Ac-DEVD-CHO-insensitive nuclear morphologic changes are inducedin intact HeLa cells after in vivo incubation with YT cell granulecontents. HeLa cells were incubated with YT cell granule contents for 1hr at 37° C. in the absence (8A & 8B) or presence (8C) of 100 μMAc-DEVD-CHO as described in Experimental Procedures. After fixation andpermeabilization, cells were stained with antibodies to PARP, as well aspropidium iodide (PI) and DAPI. Antibody staining was visualized withFYFC-goat anti-human antibodies. Merged images of antibody staining(green), PI staining (red) and DAPI staining (blue) are presented. (8A,8B): YT cell granule contents induce prominent PI-rich surface blebs,nuclear condensation and fragmentation, with a characteristicredistribution of PARP to the rim of the condensing nucleus (8A) orapoptotic bodies (8B, arrows). (8C): Granule contents induce markednuclear condensation even in the presence of Ac-DEVD-CHO (arrows); anadjacent, normal HeLa cell nucleus that has not yet undergonemorphologic change is shown for comparison (arrowhead). Note thattreatment with Ac-DEVD-CHO abolishes the formation of PI-rich surfaceblebs, nuclear fragmentation, and the redistribution of PARP. Size bar:8A: 4.4 μm; 8B: 4.0 μm; 8C: 6.6 μm.

FIGS. 9A-9B show a 2101 amino acid sequence of NuMA as found on Entrezat ACCESSION 284337; PID g284337; DBSOURCE PIR: locus A42184 SEQ ID NO:32. FIGS. 10A-10B shows a 2115 amino acid sequence of NuMA as found onEntrez at ACCESSION 107227, PID g107227; DBSOURCE PIR: locus S23647 SEQID NO: 33.

FIGS. 11A-11C show the amino acid sequence of DNA PK_(CS) as found onEntrez at ACCESSION 1362789; PID g1362789; DBSOURCE PIR: locus A57099SEQ ID NO: 34.

FIGS. 12A-12B show the amino acid sequence of PARP as found on Entrez atACCESSION 130781; PID g130781; DBSOURCE SWISS-PROT: locus PPOL_HUMAN,accession P09874 (listing only references 1 & 2 of 12) SEQ ID NO: 35.

SUMMARY OF THE INVENTION

The present invention provides autoantigenic fragments and methods fortheir use in the treatment of autoimmune disease. Also provided areassays for detecting an autoimmune condition in an animal, including thepresence of an autoimmune disease.

Caspase-mediated proteolysis of downstream substrates is a criticalelement of the central execution pathway common to all forms ofapoptosis studied to date. While cytolytic lymphocyte granule-inducedcell death activates this caspase-dependent pathway, recent studies havealso provided evidence for caspase-independent pathways in this form ofcell death. However, non-caspase substrates for granzyme B (andpotentially other granule proteases) during granule-induced cell deathhave not previously been defined. The present invention makes use of theobservation that cellular components are directly and efficientlycleaved by granule contents, including in particular granzyme B, invitro and in vivo, and that this cleavage leads to the generation ofunique autoantigenic fragments not observed during other forms ofapoptosis. This direct, caspase-independent ability of granzyme B tocleave downstream death substrates to autoantigenic fragments is anapoptotic effector mechanism which is insensitive to inhibitors of thesignaling or execution components of the endogenous apoptotic cascade.

An aspect of this invention is a composition that includes at least oneautoantigenic fragment. The autoantigenic fragment is produced by theaction of a granule enzyme on an autoantigen. In a preferred embodiment,the enzyme is a granule enzyme of CTL, NK or LAK cell granules. In amost preferred embodiment, the enzyme is granzyme B and the antigenicfragment is produced by the cleavage of the autoantigen by granzyme B ata site that is not cleaved by a caspase. In a preferred embodiment theautoantigen is DNA PK_(CS), PARP or NuMA. In a most preferredembodiment, the autoantigenic fragment is one or more of DNA-PK_(CS)from amino acid 2699 to 4096 SEQ ID NO: 34; DNA-PK_(CS) from amino acid3211 to 4096 SEQ ID NO: 34; PARP from amino acid 1 to 537 SEQ ID NO: 35;PARP from amino acid 538 to 1004 SEQ ID NO: 35; NuMA from amino acid 412to 2111 SEQ ID NO: 33 and NuMA from amino acid 1 to 1799 SEQ ID NO: 33.

An aspect of this invention is a pharmaceutical composition made withone or more purified and isolated autoantigenic fragments. In apreferred embodiment, the autoantigenic fragment has at least one endderived from granzyme B cleavage at a site in the autoantigen that isnot cleaved by a caspase. A pharmaceutically acceptable carrier is alsoincluded. In a preferred embodiment the composition includes one of moreof the following autoantigenic fragments: DNA-PK_(CS) from amino acids2699 to 4096 SEQ ID NO: 34; DNA-PK_(CS) from amino acids 3211to 4096 SEQID NO: 34; PARP from amino acid 1 to 537 SEQ ID NO: 35; PARP from aminoacids 538 to 1004 SEQ ID NO: 35; NuMA from amino acids 412 to 2111 SEQID NO: 33 and NuMA from amino acids 1 to 1799 SEQ ID NO: 33. In anotherpreferred embodiment the pharmaceutical composition includes one or moreautoantigenic fragments derived from a malignant cell.

An aspect of this invention is a method of treating a patient in need oftreatment for an autoimmune disease by administering to the patient anautoantigenic fragment of this invention. The autoimmune disease can beorgan specific, e.g., Type I diabetes mellitus, thyroiditis, myastheniagravis, primary biliary cirrhosis, or systemic in nature e.g. systemiclupus erythematosus, rheumatoid arthritis, polymyositis,dermatomyositis, Sjogrenís syndrome, scleroderma, and graft-vs.-hostdisease. In one preferred embodiment, the treatment is therapeutic. Forexample, a patient suffering from an immune disease can be administeredan autoantigenic fragment by contacting the sera of the patient with thefragment under conditions that allow the binding of autoantibodies inthe sera to bind to the fragment. In this embodiment, the level ofautoantibodies circulating in the patient can be reduced. In anotherembodiment the treatment is prophylactic. In this embodiment, a patientwho is at risk of developing an autoimmune disease is tolerized to atleast one autoantigenic fragment. Thereafter, the risk of, or severityof an autoimmune disease arising upon the later production of theautoantigenic fragment in vivo, is reduced or eliminated. In a preferredembodiment, a patient is tolerized by identifying a target tissue towhich an autoimmune disease can arise, providing at least one granuleenzyme, contacting the granule enzyme with cells from the target tissueto produce autoantigenic fragments of autoantigens present in the cells.The autoantigenic fragments are then administered to the patient totolerize the patient to the presence of the fragments. In preferredembodiments, the autoantigens can be partly or wholly purified from thecells of the target tissue. The granule enzyme can also be partly orwholly purified before contacting with the autoantigens. The enzyme canalso be made by recombinant methods. In a preferred embodiment, theautoantigenic fragments are partly or wholly purified before they areadministered to the patient. In prophylactic methods of tolerizing, theautoantigenic fragments are administered in pharmaceutically acceptablecompositions that are designed not to raise an immune response to thefragments, i.e., no immunostimmulatory adjuvants are administered withthe fragments. In a preferred embodiment, the treatment uses one or moreof the following autoantigenic fragments: DNA-PK_(CS) from amino acids2699 to 4096 SEQ ID NO: 34; DNA-PK_(CS) from amino acids 3211to 4096 SEQID NO: 34; PARP from amino acid 1 to 537 SEQ ID NO: 35; PARP from aminoacids 538 to 1004 SEQ ID NO: 35; NuMA from amino acids 412 to 2111 SEQID NO: 32 and NuMA from amino acids 1 to 1799 SEQ ID NO: 32.

An aspect of this invention is a method of treating a patient in need oftreatment for a malignancy. In a preferred embodiment, at least oneenzyme of a lymphocyte granule is contacted with the malignant cellsfrom the patient. This can produce a mixture containing autoantigenicfragments derived from the malignant cells. The fragments areadministered to the patient, preferably with an adjuvant, to stimulatean immune response against the malignant cells.

An aspect of this invention is an assay for the detection of anautoantigenic fragment in a patient. In one embodiment, the presence orabsence of the fragment in a patient sample is an indication of thepresence or absence of an autoimmune condition in the patient. In apreferred embodiment, a sample from the patient is contacted with anantibody that specifically binds to a cryptic epitope of anautoantigenic fragment. Preferably, the fragment has at least oneterminus derived from the cleavage of an autoantigen by granzyme B at asite that is not cleaved by a caspase. The presence or absence of thebinding of the antibody to the autoantigenic fragment is then assessedas an indication of the presence or absence of an autoimmune conditionin a patient. In an alternative embodiment, the detection of an antibodythat binds an autoantigenic fragment is an indication of the presence orabsence of an autoimmune condition in the patient. In this embodiment asample from the patient is contacted with an autoantigenic fragmenthaving at least one terminus derived from cleavage by a granule enzyme.Detection of the presence or absence of the binding of an antibody inthe sample to the autoantigenic fragment is an indication of thepresence or absence of an autoimmune condition in the patient.

An aspect of this invention is a method of making an autoantigenicfragment from an autoantigen. In a preferred embodiment, one isolatescells containing at least one autoantigen and contacts the cells with alymphocyte granule enzyme to produce a mixture containing at least oneautoantigenic fragment. In a further embodiment one isolates at leastone autoantigenic fragment from the mixture. In a preferred embodimentone purifies at least one autoantigen and contacts the purifiedautoantigen with granzyme B. In a further preferred embodiment, onepurifies one or more of the following autoantigens for contacting withgranzyme B: DNA-PK_(CS), PARP and NuMA. In each embodiment the granuleenzyme can isolated from the granules of a lymphocyte, e.g., a cytotoxicT lymphocyte (CTL), a natural killer cell (NK), a lymphokine activatedkiller cell (LAK) or cells of the YT cell line.

In all aspects of this invention, granzyme B can be used in particularembodiments. The enzyme can be purified from the granules of granulecontaining lymphocytes or can be prepared by recombinant techniques.

Definitions:

As used herein, “treatment” includes the therapeutic or prophylacticapplication of a composition to a patient. A treatment can prevent,moderate or cure a disease in the patient. A disease is moderated in apatient when the treatment lessens the severity or frequency of at leastone symptom associated with the disease. A treatment can moderate adisease by: (1) prophylactic administration of a composition to apatient free of a disease to lessen the impact of at least one symptomof the disease when it does occur or (2) therapeutic administration to apatient having a disease to lessen at least one symptom of the disease.

As used herein, a “patient” is an animal, particularly including ahuman.

As used herein, an “autoimmune condition” is the presence of, or thepredisposition for the development of, an autoimmune disease or anautoimmune response in a patient.

As used herein, an “autoantigen” is a cellular molecule and usually is aprotein. An autoantigen is typically not antigenic because the immunesystem is tolerized to its presence in the body under normal conditions.An autoantigen will typically include at least one cryptic epitope. Anautoantigen can be produced by natural cells, using recombinant methods,or through chemical synthesis, as appropriate.

As used herein, an “autoantigenic fragment” is a degradation product ofan autoantigen. An autoantigenic fragment is antigenic because theimmune system is not tolerized to its presence in the body.Autoantigenic fragments usually display cryptic epitopes to the immunesystem. An autoantigenic fragment can be produced by natural cells,through the action of at least one granule enzyme in a cellular or cellfree system, using recombinant methods, or through chemical synthesis,as appropriate.

As used herein, an “autoantibody” is an antibody produced by the immunesystem of an animal in response to the present of an autoantigenicfragment. An autoantibody can bind to the autoantigenic fragment, theautoantigen from which the fragment is derived, or both.

As used herein, a “granule containing lymphocyte” is meant to includeall lymphocytes that contain granules. In particular, the term is usedto include the family of cell types sometimes referred to as cytotoxiclymphocytes, to include cell lines derived from these cells and toinclude cytotoxic lymphocyte-like cell lines, preferably the YT cellline. Preferred cells are the granule containing lymphocytes known inthe art as cytotoxic T lymphocytes (CTL), natural killer cells (NK) andlymphokine activated killer cells (LAK).

As used herein, a “lymphocyte granule enzyme” or “granule enzyme” is anenzyme that is found in the granules of a granule containing lymphocyte.A granule enzyme can be purified from a lymphocyte granule by methodscommonly employed in the art of protein purification. Additionally, agranule enzyme can be prepared by cloning the gene for the enzyme andthe enzyme then is prepared using methods commonly used in theproduction of recombinant enzymes.

As used herein, “purified” and/or “isolated” are used interchangeably tostand for the proposition that the protein(s) and polypeptide(s), orrespective fragment(s) thereof in question has been removed from its invivo environment. A protein or fragment thereof is considered “purified”and/or “isolated” when it is obtained at a concentration at least aboutfive-fold to ten-fold higher than that found in nature. A protein orfragment thereof is considered substantially pure if it is obtained at aconcentration of at least about 100-fold higher than that found innature. A protein or fragment thereof is considered essentially pure ifit is obtained at a concentration of at least about 1000-fold higherthan that found in nature. A protein is sometimes referred to as partlypurified if it is at least purified or isolated but it is notessentially pure. A chemically synthesized protein is considered to besubstantially purified when purified from its chemical precursors. Apurified or isolated protein can be manipulated by the skilled artisan,such as but not limited to obtaining the protein or protein fragment inquantities that afford the opportunity to generate polyclonalantibodies, monoclonal antibodies, amino acid sequencing, and peptidedigestion. Therefore, the autoantigenic fragments claimed herein can bepresent in cell lysates or in a substantially or essentially pure form.

Abbreviations:

Ac-DEVD-CHO, N—(N-Ac-Asp-Glu-Val)-3-amino-4-oxobutanoic acid SEQ ID NO:1; Ac-YVAD-CHO, N—(N-Ac-Tyr-Val-Ala)-3-amino-4-oxobutanoic acid SEQ IDNO: 26; CTL, cytotoxic T lymphocytes; DNA-PK_(CS), DNA-dependent proteinkinase catalytic subunit; LAK cells, lymphokine-activated killer cells;NK cells, natural killer cells; NuMA, nuclear mitotic apparatus protein;PARP, poly(ADP-ribose)polymerase; PI, propidium iodide

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides autoantigenic fragments and methods fortheir use in the treatment of autoimmune disease. Also provided areassays for detecting an autoimmune condition in an animal, including thepresence of an autoimmune disease.

The present invention makes use of the discovery that a class ofpreviously unrecognized autoantigenic fragments is generated during theform of apoptosis triggered by the action of the contents of lymphocytegranules on cells. In particular, enzymes found in lymphocyte granulesare discovered to cleave proteinaceous cellular autoantigens to yieldpreviously unrecognized autoantigenic fragments. Granzyme B is found tobe an important granule enzyme in this generation of autoantigenicfragments. This enzyme was previously known to cleave some of thepro-caspase enzymes to yield active caspases. Granzyme B is now found todirectly cleave certain of the substrates of the caspases at differentsites to produce novel autoantigenic fragments.

The present invention provides a method of producing granzyme generatedautoantigenic fragments in vitro in several ways. In a preferredembodiment, purified granzyme B can be contacted with purifiedsubstrates to produce the autoantigenic fragments. Partly or whollypurified enzyme can also be used on partially or wholly purifiedsubstrates or cellular lysates containing the substrates. In this case,the autoantigenic fragments can be purified after cleavage of thesubstrates. Additionally, the contents of granules can be used toproduce autoantigenic fragments of cellular components, including theautoantigenic fragments of caspase substrates created by the action ofgranzyme B, by application of granule contents, or purified substratesor tissue samples isolated from a patient. In some embodiments, thecells of the tissue can be disrupted by lysis or mechanical breakage torelease the contents of the cells before contacting the cells with thecontents of the granules.

The autoantigenic fragments provided herein can be used in a treatmentto tolerize a patient to the presence of the autoantigenic fragments.Once tolerized, the patient would not develop an autoimmune diseaseassociated with the later appearance of the fragments in the patient.Tolerizing strategies involve purification of relevant autoantigenicfragments in a non-aggregated form. Low doses of the fragments areinjected in pharmaceutically acceptable carriers, preferably without anadjuvant to induce low-zone tolerance.

The present invention also provides a method of generating an autoimmuneresponse against certain cells in a patient. For example, if it isdesirable to generate an autoimmune response against malignant cells ina patient, one can isolate a sample of the cells from the patient andcontact the cells with the contents of granules isolated from granulecontaining lymphocytes. The action of the granule contents on the cellscan produce autoantigenic fragments therefrom. In some embodiments, themalignant cells can be disrupted by lysis or mechanical breakage torelease the contents of the cells before contacting the cells with thecontents of the granules. In any case, the resulting mixture or purifiedcomponents therefrom can be administered to the patient. Because theautoantigenic fragments produced in this way are the same as thoseproduced in vivo by the action of granule containing lymphocytes,including e.g., CTLs, NK and LAK cells, on the malignant cells, theimmune system is thereby stimulated to generate a response against themalignant cells. Therefore, the present invention provides a method toheighten or stimulate the natural immune system processes to act againstparticular types of cells such as malignant cells.

The autoantigenic fragments useful in the invention described hereindisplay cryptic epitopes. These epitopes are revealed to the immunesystem after cleavage of the precursor protein by the enzymes containedin granules to yield the autoantigenic fragments. There is a persuasivebody of literature that reports that the highly specific humoral immuneresponse to autoantigens in autoimmune disease is T cell-dependent, andthat flares in autoimmune disease result when this primed immune systemis rechallenged with a self-antigen (reviewed in Burlingame, R. W., etal., 1993; Diamond, B., et al., 1992; Radic, M. Z. and M. Weigert.1994). However, the mechanisms responsible for initiation of the primaryimmune response to these molecules, and for subsequently stimulating thesecondary response to targeted antigens, have not been completelyelucidated (Bach, J. F. and S. Koutouzov. 1997; Sercarz, E. E. and S. K.Datta. 1994). Several studies report that a potential for T cellautoreactivity resides in the immunological non-equivalency of differentareas of self-molecules, since tolerance is only induced to dominantdeterminants which are generated and presented at suprathresholdconcentrations during natural processing of whole protein antigens(reviewed in (Sercarz, E. E., et al., 1993; Lanzavecchia, A. 1995).Those determinants which are not generated at all, or are generated atsubthreshold levels during antigen processing (termed cryptic), do nottolerize T cells. Thus, potentially autoreactive T cells recognizingthis cryptic self epitope are allowed to persist.

The mechanisms through which autoimmunity can arise when normallycryptic determinants become visible to the immune system have receivedincreased attention. Several experimental systems have now providedevidence that the balance of dominant versus cryptic epitopes in a selfmolecule can be profoundly influenced by forces which alter the‘immunological’ structure of molecules (Lanzavecchia, A. 1995). Examplesinclude the revelation of cryptic epitopes through novel cleavage(Bockenstedt, L. K., et al., 1995; Mamula, M. J. 1993), or throughaltered conformation induced by high affinity ligand binding (e.g. to anantibody or receptor molecule (Salemi, S., et al., 1995; Simitsek, P.D., et al., 1995; Watts, C. and A. Lanzavecchia. 1993). The unique,high-titer autoantibody responses that characterize different autoimmunediseases can therefore be viewed as the immunologic impression of theinitiating events that revealed suprathreshold concentrations ofnon-tolerized structure in a pro-immune context, thus satisfying thestringent criteria for initiation of a primary immune response(Casciola-Rosen, L. and A. Rosen. 1997).

The alteration of the structure of autoantigens during apoptosis is animportant feature which underlies targeting of specific molecules by theimmune system. Understanding whether and how the structure of thoseautoantigens are cleaved during apoptosis, provides insights into therole of apoptosis in initiation of the autoimmune response. One suchmechanism involves the action of the enzymes in contents of granulesfound in granule containing lymphocytes and is described herein. Thecontents of these granules are shown to act to reveal cryptic epitopesby cleaving autoantigens to autoantigenic fragments. In a preferredmethod, granzyme B cleaves autoantigenic proteins to create preferredautoantigenic fragments that display cryptic epitopes to the immunesystem.

Recent reports using caspase inhibitors have emphasized the contributionof caspase-independent pathway(s) when target cell death is induced bycytotoxic lymphocyte granule exocytosis (Sarin et al., 1997; Talanian etal., 1997). The results presented here demonstrate that several of thedownstream substrates cleaved by the caspase family of proteases duringapoptosis are also directly and efficiently cleaved by granzyme B bothin vitro and in target cells undergoing lymphocyte granule-inducedcytotoxicity. This confirms the existence of efficientcaspase-independent proteolytic pathways during this form of cell death.

Not all downstream substrates of caspase-3 are cleaved by granzyme Bwith similar efficiency. In the case of PARP and U1-70 kDa, cleavageefficiency by caspase-3 exceeds that by granzyme B by more than 200fold, while NuMA and DNA-PK_(CS) are cleaved with similar efficienciesby granzyme B and caspase-3 (Table I). The relative cleavage efficiencyof a given substrate by the two proteases appears to account for theresults observed when caspase-3 activity is inhibited in intact cells.Thus, while PARP cleavage during granule-mediated cytotoxicity isinhibited by Ac-DEVD-CHO, cleavage of DNA-PK_(CS) and NuMA are onlyminimally affected. It is therefore believed that caspase-independentcomponents of granule-mediated death are generated through altering thefunction of those downstream substrates that are efficiently cleaved bygranzyme B (and potentially other granule proteases).

The results presented herein are consistent with recent studies in whichgranzyme B-induced proteolysis of PARP and U1-70 kDa (both inefficientdirect substrates for granzyme B) in an intact cell model was wellinhibited by caspase inhibitors (Talanian et al., 1997). The resultsdiffer from a previous study which failed to demonstrate uniqueDNA-PK_(CS) fragments during CTL-induced target cell death, or bygranzyme B in vitro (Song et al., 1996a). Different antibodies toDNA-PK_(CS) used in the two studies may account for the failure of theprevious study to find that a unique 100 kDa C-terminal fragment isgenerated by granzyme B.

The results indicate that direct and efficient cleavage of NuMA andDNA-PK_(CS) by granzyme B achieves functional effects similar to thosecaused by caspase cleavage during other forms of apoptosis. This issupported by the demonstration that the cleavage of DNA-PK_(CS) bygranzyme B completely abrogates its kinase activity (FIG. 3). Thisgranzyme B-mediated cleavage of DNA-PK_(CS) differs fromcaspase-3-mediated cleavage of this substrate in several ways: (i) Thepresence of DNA ends renders the granzyme B-mediated cleavagesignificantly less efficient, while DNA ends are required for efficientcleavage of DNA-PK_(CS) by caspase-3 (Casciola-Rosen et al., 1996; Songet al., 1996b) (ii) While caspase-3 cleavage of DNA-PK_(CS) decreasesits kinase activity to approximately 60% of control levels(Casciola-Rosen et al., 1996), cleavage by granzyme B completelyabrogates kinase activity. These data indicate that granzyme B cleavesand fully inactivates DNA-PK_(CS) very early during granule-mediatedcytotoxicity, before significant internucleosomal DNA cleavage hasoccurred, and thus before caspase-3 can efficiently cleave DNA-PK_(CS).Once abundant DNA ends have been generated, cleavage by caspase-3 shouldpredominate (with the resulting fragments possessing residual kinaseactivity).

Although the functional consequences of substrate cleavage by caspasesduring apoptosis in vivo is not known for most substrates, recentreports demonstrate that cleavage can activate critical pro-apoptoticactivities (e.g. activation of DNA fragmentation factor by caspase-3(Liu et al., 1997)). In other cases, reports suggest that substratecleavage might disable important structural and homeostatic functions(Casciola-Rosen et al., 1996; Ghayur et al., 1996). It is thereforereported that the ability of cytolytic lymphocyte granule proteases todirectly cleave some caspase substrates underlies thecaspase-independent component of the death pathway induced by thesecells (Sarin et al., 1997). The ability of the lymphocytegranule-induced cytotoxicity pathway to generate a novel form of nuclearcondensation even in the absence of caspase activity is consistent withthis theory.

Studies on macromolecular and tetrapeptide substrates provide insightsinto the substrate specificity of granzyme B. For example, the directcleavage of PARP by granzyme B occurs at VGPD⁵³⁷-S⁵³⁸ SEQ ID NO: 35(Froelich et al., 1996a), while processing of several caspase precursorsoccurs at IETD sites (Fernandes-Alnemri et al., 1996; Ramage et al.,1995; Srinivasula et al., 1996; Yamin et al., 1996). The studiespresented here demonstrate that granzyme B cleaves a macromolecularsubstrate (DNA-PK_(CS)) at VGPD-F²⁶⁹⁹ SEQ ID NO:34, a site consistentwith the tetrapeptide substrate specificity of granzyme B defined usinga combinatorial tetrapeptide library (Thornberry et al., 1997).Interestingly, granzyme B also directly cleaves this substrate at anearby DEVD-N²⁷¹³ SEQ ID NO: 34, suggesting that the determinants ofmacromolecular substrate specificity are more complex than thosecontained in the tetrapeptides tested.

Using the fragment sizes of NuMA and DNA-PK_(CS) generated by granzymeB, the known epitope specificity of several of the antibodies used, andthe known substrate specificity of granzyme B, these studies indicatethat the most likely granzyme B cleavage site generating the 100 kDaC-terminal fragment of DNA-PK_(CS) is VDQD³²¹⁰-G³²¹¹ SEQ ID NO:34.Similarly, the most likely granzyme B cleavage site in NuMA occurs atVLGD⁴¹¹-V412 SEQ ID NO: 32. Several studies have addressed the cellbiology of granzyme B during perforin/granzyme B-induced apoptosis(Froelich et al., 1996b; Shi et al., 1997; Trapani et al., 1996). Thesestudies reported that granzyme B autonomously enters the cytoplasm oftarget cells. That event alone does not induce target cell apoptosis. Inthe presence of perforin, however, apoptosis is induced in target cells.That event is accompanied by the rapid enrichment of granzyme B innuclei and nucleoli of target cells (Jans et al., 1996; Pinkoski et al.,1996; Trapani et al., 1996). It is therefore of importance that thegranzyme B substrates described here are nuclear proteins. Thesesubstrates function in both structural and homeostatic pathways whichare impacted by the caspases during apoptosis. The observation that acomponent of granule-induced cell death is caspase-independent, takentogether with the ability of caspases and granzyme B to efficientlycleave a common subset of downstream substrates at different sitesduring granule-induced cytotoxicity, highlights the importance ofproteolysis of those common substrates in generating the apoptoticphenotype. Table II).

In the light of several descriptions of viral or endogenous caspaseinhibitors (Beidler et al., 1995; Bump et al., 1995; Irmler et al.,1997; Thome et al., 1997; Xue and Horvitz, 1995), as well as long-livedcells or tumor cells which express low levels of specific caspase familymembers (Krajewska et al., 1997; Krajewski et al., 1997), thecaspase-independent activity of granzyme B provides the host withapoptotic effector mechanisms that are insensitive to inhibitors of thesignaling or execution components of the apoptotic cascade.

Many of the downstream caspase substrates described to date areautoantigens targeted in human systemic autoimmune diseases(Casciola-Rosen et al., 1995; Casciola-Rosen et al., 1994;Casciola-Rosen et al., 1996; Casiano et al., 1996; Greidinger et al.,1996). The present demonstration that several of these substrates arealso directly cleaved by granzyme B, generating unique fragments notgenerated during any other form of apoptosis studied to date,demonstrates how non-tolerized determinants of autoantigens can berevealed during certain forms of CTL-mediated apoptotic death.

In the experimental results presented herein, it is demonstrated thatDNA-PK_(CS) and NuMA are directly cleaved by granzyme B, both in vitroand in cells undergoing granule-induced cytotoxicity. Although theefficiency of cleavage of these substrates is similar to those observedfor caspase 3-mediated cleavage, the fragments generated by the 2proteases are distinct. Since caspases appear to initiate apoptosis byaltering the function of downstream substrates (either by decreasing thefunction of the intact substrate, or by generating fragment(s) withpro-apoptotic activity), it is believed that direct cleavage of caspasesubstrates by granzyme B during cytotoxic lymphocyte granule-inducedapoptosis plays an important role in caspase-independent target celldeath. The ability of the contents of cytotoxic lymphocyte granules tobypass the requirement for caspases in the death pathway may guaranteethe demise of target cells whose caspase pathway is incomplete or understrict endogenous or exogenous regulatory control.

The action of granzyme B in producing particular autoantigenic fragmentsfrom particular autoantigens is exemplified herein. However, the generalunderstanding of the role of granule proteases as described andexemplified in the granzyme B model system allows one to generate theseand other autoantigenic fragments. The autoantigenic fragments producedcan be used in the preparation of pharmaceutical compositions, fortreating patients at risk for or suffering from autoimmune diseases andcancer, and in assays for assessing the presence or absence of anautoimmune condition in a patient.

The following examples are presented by the way of illustration and,because various other embodiments will be apparent to those in the art,the following examples are not to be construed as a limitation on thescope of the invention.

EXAMPLE 1

General Materials and Methods

Materials. Purified DNA-dependent protein kinase (DNA-PK) and SP1 werepurchased from Promega (Madison, Wis.). ATP was purchased from Fluka(Ronkonkoma, N.Y.), and ³²P-ATP was from Du Pont/NEN (Wilmington, Del.).Ac-DEVD-CHO and Ac-YVAD-CHO were manufactured by Merck (Rahway, N.J.).Caspase-3 was purified as described (Nicholson et al., 1995). Patientsera were used to immunoblot the nuclear mitotic apparatus protein(NuMA), poly(ADP-ribose) polymerase (PARP) and DNA-PK_(CS)(Casciola-Rosen et al., 1995; Greidinger et al., 1996). Monoclonalantibodies can be made by methods known in the art. Two differentmonoclonal antibodies, designated 18-2 and 25-4 (kind gifts from Dr. TimCarter, St. Johns University, Jamaica, N.Y.) were also used to detectDNA-PK_(CS) by immunoblotting (see Table II for a summary of theantibodies used to detect DNA-PK_(CS) and its cleaved fragments). Rabbitpolyclonal antibodies to caspases were raised against the large subunitsof caspase-3 and caspase-7, using methods commonly known in the art.Immunoblotted proteins were detected using the SUPERSIGNAL™ substratesystem (Pierce, Rockford, Ill.), according to the manufacturer'sinstructions.

In vitro cleavage of purified DNA-PK_(CS) and [³⁵S]methionine-labeledcaspase-3 precursor, caspase-7 precursor, PARP, and NuMA. cDNAs forcaspase-3, caspase-7, NuMA and PARP were used to drive the synthesis of[³⁵S]methionine-labeled proteins by coupled transcription/translation inrabbit reticulocyte lysates. For all purified substrates, cleavagereactions were performed in buffer consisting of 50 mM Hepes pH 7.4, 10%sucrose and 5 mM DTT in the presence of the granzyme B concentrationsindicated in FIG. 1. After incubation at 37° C. for 15 min, reactionswere terminated and samples were electrophoresed on 10% (DNA-PK_(CS),NuMA), 12% (PARP) or 15% (caspases 3 and 7) SDS-polyacrylamide gels.Radiolabeled proteins and their fragments were visualized byfluorography. Intact and cleaved DNA-PK_(CS) were visualized byimmunoblotting with monoclonal antibody 18-2 (Casciola-Rosen et al.,1995).

Calculation of catalytic constant values. Catalytic constant(k_(cat)/k_(m)) values were calculated essentially as described(Casciola-Rosen et al., 1996). Briefly, subsaturating substrateconcentrations were used in each in vitro reaction, and productappearance was assumed to be a first order process. Substrate andproduct bands on autoradiograms were scanned by densitometry. Severalappropriate densitometry systems are available, e.g. PDI DiscoverySystem, with Quantity One Software, Protein Databases, Inc., (HuntingtonStation, N.Y.). kcat/Km values were calculated by fitting thedose-response data to the first order rate equation: percent substratecleavage=100*(1^(-e-)((kcat*[E]/Km)*time)).

In vitro cleavage of endogenous DNA-PK_(CS), NuMA, and PARP in HeLalysates. Control HeLa lysates were prepared using methods commonlyapplied in the art, as described in Casciola-Rosen et al., 1994. 12.5 nMpurified granzyme B or 105 pM purified caspase-3 were then added to thelysates, in the absence or presence of 100 nM Ac-DEVD-CHO orAc-YVAD-CHO. The mixtures were incubated for 15 min on ice to facilitatebinding of the inhibitors to proteases prior to performing cleavagereactions at 37° C. for 60 min. After electrophoresing the samples on10% SDS-polyacrylamide gels containing 0.087% bisacrylamide, the intactproteins and their cleaved fragments were visualized by immunoblotting.

Determination of granzyme B cleavage sites in DNA-PK_(CS) by PI Aspmutagenesis. A partial cDNA clone for DNA-PK_(CS), encoding Met²⁵⁶⁶through Leu²⁹²⁸ (the region containing the caspase-3 cleavage site aswell as the putative granzyme B site), was amplified byreverse-transcriptase PCR from HeLa cell poly(A)+ RNA using primerscontaining 5′ EcoRI and 3′ XbaI restriction enzyme-adapters. Afterligation into the corresponding restriction sites of pBluescript II SK+(Stratagene), this clone was used as template for mutagenesis byoverlap-extension PCR to generate clones containing D²⁶⁹⁸A (P1 of theputative granzyme B site) and D²⁷¹²A (P1 of the known caspase-3 site)modifications. [³⁵S]-Radiolabeled polypeptides were generated by coupledin vitro transcription/ translation, and then incubated with eitherrecombinant caspase-3 (8 nM) or purified YT cell-derived granzyme B (8nM) for 60 min at 37° C. in a buffer composed of 50 mM Hepes/KOH (pH7.0), 10% (w/v) sucrose, 2 mM EDTA, 0.1% (w/v) CHAPS, 5 mMdithiothreitol. The resulting cleavage products were resolved onSDS-polyacrylamide gels (10-20% gradient gels) and visualized byfluorography.

In vivo cleavage of endogenous DNA-PK_(CS), NuMA and PARP during YT cellgranule content-induced cytotoxicity of intact K562 cells. Intactcytoplasmic granules were purified from YT cells, and the granulecontents were isolated using known methods (Tschopp, 1994). Some ofthese preparations were used for the further purification of granzyme B(Tschopp, 1994). Purity of the protease was confirmed by silver stainingof overloaded SDS-polyacrylamide gels. The cytotoxic effects of YTgranule contents were determined as follows: Jurkat T cells or K562cells were radiolabeled with 100 μCi/ml [⁵¹Cr] sodium chromate, washed,resuspended in Ca²⁺-free HBSS, and then incubated with granule contents(0-5 μl) and 1 mM CaCl₂ for increasing times. The percentage specific[⁵¹Cr] was calculated using the following formula: % specificlysis=[(sample cpm−spontaneous cpm)/(maximum cpm−spontaneous cpm)]×100.In vivo experiments to assay the effect of YT cell granule contents onDNA-PK_(CS), NuMA and PARP in intact K562 cells were performed asfollows: K562 cells were washed twice with PD (2.7 mM KCl, 1.5 mMKH₂PO₄, 137 mM NaCl, 8 mM Na₂HPO₄), then resuspended at 1.7×10⁷ cells/mlin PD in the absence or presence of 100 μM Ac-DEVD-CHO, and incubated at37° C. for 30 min. Aliquots containing 3.4×10⁵ cells were incubated fora further 90 min at 37° C. with 1 mM EDTA or 1 mM Ca²⁺ and 2 μl of YTgranule contents (which induces 20-40% specific chromium release in 60min, and characteristic internucleosomal DNA degradation. The totalreaction volume of each sample was 30 μl. The reactions were terminatedby boiling in SDS gel buffer and samples were electrophoresed andimmunoblotted as described above. Experiments were performed using 2different preparations of granule contents and 4 different cell types(K562, Jurkat, HeLa, primary human myoblasts).

Confocal Immunofluorescence microscopy. Morphologic experiments wereperformed on HeLa cells grown on No. 1 glass coverslips. Coverslips werewashed three times with ice-cold HBSS without Ca²⁺, prior to incubation(4° C., 30 min) with 25 μl of HBSS minus Ca²⁺ containing 0.8 μl of YTcell granule contents (see above), in the presence or absence of 200 μMAc-DEVD-CHO. 25 μl of HBSS containing 2 mM CaCl₂ was then added to eachcoverslip (mixed well by repeated, gentle aspiration), followed byincubation in a humidified chamber at 37° C. for 60 min. The cells werethen fixed in 4% paraformaldehyde (4° C., 5 min), permeabilized withacetone (4° C., 15 sec), and stained sequentially with antibodies toPARP or NuMA, propidium iodide and DAPI as described (Casciola-Rosen etal., 1994a). Coverslips were mounted on glass slides with Permafluor(Lipshaw, Pittsburgh, Pa.), and confocal microscopy was performed on ascanning confocal microscopy system (LSM 410, Carl Zeiss, Inc.,Thornwood, N.J.).

LAK cell-mediated cytotoxicity. LAK cells were obtained by culturinghuman PBMCs for 4 days in LAK medium (RPMI supplemented with 10 mM HepespH 7.4, L-glutamine, 2% autologous plasma), and 1000 Cetus units/ml ofhrIL-2 (Chiron Therapeutics, Emeryville, Calif.) (Topalian et al.,1989). Fas-negative target cells (K562 erythroleukemia cells) wereresuspended at 1.3×10⁶ cells/ml in LAK medium in the presence or absenceof 100 μM Ac-DEVD-CHO, and incubated at 37° C. for 60 min, prior toco-incubation with LAK effector cells (effector:target ratio of 5:1) for4 h. After 2 washes with PD, cells were lysed and boiled in SDS samplebuffer, and PARP, DNA-PK_(CS) and NuMA were assayed by immunoblotting asdescribed above.

Kinase assay. Kinase assays were performed on intact DNA-PK_(CS) orDNA-PK_(CS) that had first been cleaved by granzyme B as follows.Reaction mixtures containing 10 mM Hepes pH 7.4, 2 mM MgCl₂, 10 mM KCl,2.7 mM DTT, and 50 ng DNA-PK_(CS) in the absence or presence of 12.5 nMpurified granzyme B, were incubated for 13.5 min at 37° C. Kinasereactions were subsequently initiated by adding 100 ng SP1 and 150 μMATP containing 1.5 μCi [³²P]-ATP (3000 Ci/mmol), in the absence orpresence of 10 μg/ml sheared herring sperm DNA (Promega). Samples wereincubated at 37° C. for 10 min (well within the linear range of theassay, data not shown), before terminating the reactions by adding SDSgel buffer and boiling. After electrophoresing the samples on 8%SDS-PAGE, SP1 phosphorylation was detected by autoradiography, andquantitated by densitometry. Cleaved status of the kinase was confirmedin parallel by immunoblotting.

EXAMPLE 2

DNA-PK_(CS) and NuMA are Very Efficient Substrates for Purified GranzymeB.

Granzyme B has previously been reported to cleave the precursors ofseveral caspases (including caspases 3, 7 and 10), resulting inactivation of their proteolytic activity. The catalytic efficiency ofcleavage of these substrates by granzyme B serves as a useful standardagainst which granzyme B-mediated cleavages of other substrates can becompared. Purified [³⁵S]methionine-labeled precursors of caspase-3 andcaspase-7, or THP.1 cytosols (containing these precursor proteases) wereincubated in vitro with increasing concentrations of purified granzymeB. The dose-response data obtained (FIG. 1) was used to calculatecatalytic constant (k_(cat)/K_(m)) values of 1.8±0.6×10⁵M⁻¹s⁻¹(radiolabeled substrate) and 1.9±0.1×10⁵ M⁻¹s⁻¹ (immunoblotting) forcaspase-7, and 3.6±1.0×10⁴ M⁻¹s⁻¹ (radiolabeled substrate) and2.3±0.4×10⁴M⁻¹s⁻¹ (immunoblotting) for caspase-3 (Table I). Thus,granzyme B cleaves caspase-7 approximately 6 fold more efficiently thancaspase-3, consistent with previous reports (Talanian et al., 1997).

It was then determined whether any of the downstream substrates known tobe cleaved by caspases during apoptosis were also directly cleaved bygranzyme B, and the efficiency of cleavage of each substrate by the twoproteases was compared. Purified DNA-PK_(CS) was very efficientlycleaved by granzyme B in the absence of added DNA(k_(cat)/K_(m)=2.5±0.8×10⁶M⁻¹s⁻¹, see FIG. 1 and Table I), making it thebest substrate for granzyme B described to date, with a cleavageefficiency two orders of magnitude better than that described forgranzyme B-mediated cleavage of the caspase-3 precursor. When similarexperiments were performed in the presence of 10 μg/ml DNA, DNA-PK_(CS)cleavage was decreased by approximately 90%.

Caspase-3-mediated cleavage of DNA-PK_(CS) was also extremely efficient(k_(cat)/K_(m) value=7.5±0.8×10⁶M⁻¹s⁻¹). In this case, efficientcleavage was only obtained in the presence of DNA ((Casciola-Rosen etal., 1995), and Table 1). NuMA, a nuclear matrix protein that is cleavedin apoptotic cells by an unidentified protease with features of thecaspase family, was also very efficiently cleaved by granzyme B(k_(cat)/K_(m) value=5.4±1.4×10⁵M⁻¹s⁻¹, see Table I). The efficiency ofthis cleavage was one order of magnitude greater than that observed forgranzyme B-mediated processing of the caspase-3 precursor. NuMA was alsoefficiently cleaved by purified caspase-3, with a k_(cat)/K_(m) valueof=5.0±1.0×10⁵M⁻¹s⁻¹. (Table I). In contrast to DNA-PK_(CS) and NuMA(where granzyme B- and caspase-3-mediated cleavages are similarlyefficient), PARP was a relatively poor substrate for granzyme B, with ak_(cat)/K_(m) value (2.3±1.8×10⁴ M⁻¹s⁻¹) that is approximately 200 foldlower than that for caspase-3 (see Table I). Granzyme B was also a poorcatalyst for cleaving U1-70 kDa, with k_(cat)/K_(m) values <10³ M⁻¹s⁻¹.The efficiency of substrate cleavage by granzyme B is therefore similarto caspase-3 for some substrates (e.g. DNA-PK_(CS) and NuMA), while itis more than 2 orders of magnitude less efficient for others (e.g. PARPand U1-70 kDa).

EXAMPLE 3

Different Substrate Fragments are Detected After Cleaving Autoantigensin Vitro with Granzyme B or Caspase-3.

To directly compare the fragments generated by granzyme B and caspase-3,purified DNA-PK_(CS), in vitro translated [³⁵S]methionine-labeled PARP,and endogenous substrates (NuMA and DNA-PK_(CS) in HeLa cell lysates)were incubated with protease and electrophoresed in adjacent lanes. Whengranzyme B was used to cleave DNA-PK_(CS), fragments of 100 kDa and 250kDa were generated, (detected by immunoblotting using antibodiesrecognizing the C-terminus or N-terminus of DNA-PK_(CS), respectively)(FIG. 2, lanes 2 & 5; and Table II). In contrast, caspase-3 cleavageyielded a 150 kDa C-terminal fragment (FIG. 2, lane 3) and a 250 kDaN-terminal fragment (FIG. 2, lane 6).

Granzyme B-mediated cleavage of NuMA generated a novel fragmentmigrating at 175 kDa on SDS-PAGE, which was distinct from the 185 kDafragment detected after cleavage with caspase-3 (FIG. 2, lanes 7-9).Similarly, novel fragments of PARP migrating at 72, 62 and 42 kDa weredetected after incubation with granzyme B; these differed from the 89and 24 kDa fragments generated by caspase-3-mediated cleavage of PARP(FIG. 2, lanes 10-12 and Table II). Granzyme B therefore directlycleaves several of the downstream substrates of caspase-3 in vitro. Inall cases, the fragments generated by granzyme B differ from thosegenerated by caspase-3.

EXAMPLE 4

Kinase Activity of DNA-PK_(CS) is Abolished by Granzyme B Cleavage.

To determine the effect of granzyme B-mediated cleavage on the kinaseactivity of DNA-PK_(CS), the ability of intact and cleaved DNA-PK_(CS)to phosphorylate the SP1 transcription factor was quantitated. Thekinase activity of DNA-PK_(CS) was entirely DNA-dependent (FIG. 3, lanes1 and 2). When DNA-PK_(CS) was pretreated with 12.5 nM granzyme B for13.5 min at 37° C. (which cleaves DNA-PK_(CS) (FIG. 1)), followed by theaddition of DNA, [³²P]ATP and SP1 (which initiate the phosphorylationreaction), kinase activity was entirely abolished (FIG. 3, lane 3).Whether any of the fragments generated by granzyme B-mediated cleavageof DNA-PK_(CS) have novel activity(ies) in addition to the autoantigenicreactions demonstrated herein remains to be determined.

EXAMPLE 5

Granzyme B Induces Novel Fragments of DNA-PK_(CS) and NuMA in CellLysates.

Previous studies reported that the cleavage of PARP that occurs inlysates of COS cells expressing granzyme B can be almost completelyinhibited by the caspase inhibitor, Ac-DEVD-CHO (Darmon et al., 1995).Because this compound is not an inhibitor of granzyme B, thisobservation suggests that this cleavage is mediated solely by caspaseswhich have been activated by granzyme B in these extracts. Theobservation that PARP itself is cleaved by granzyme B approximately 200fold less efficiently than by caspase-3 further supports this conclusion(see Table I). Since NuMA and DNA-PK_(CS) are cleaved with similarefficiencies by caspase-3 and granzyme B, it was of interest todetermine whether these substrates are cleaved directly by granzyme B incell lysates that contain caspase precursors. Therefore, an in vitroassay system was established in which cleavage of endogenousDNA-PK_(CS), NuMA or PARP was monitored after addition of purifiedcaspase-3 or granzyme B. The assay was conducted in the presence orabsence of the caspase inhibitor, Ac-DEVD-CHO.

Incubation of control extracts at 37° C. for 60 minutes resulted in nosignificant cleavage of DNA-PK_(CS) or PARP, but was associated withminimal cleavage of NuMA, leading to the production of minor fragmentsof 185 and 187 kDa (FIG. 4, lane 1). When purified caspase-3 was addedto control extracts, caspase-3-specific fragments of PARP (89 kDa), NuMA(185 kDa) and DNA-PK_(CS) (250 kDa N-terminal fragment, 150 kDaC-terminal fragment) were generated (FIG. 4, lane 2). As demonstratedabove, substrate fragments generated by the activity of caspase-3 areentirely abolished by 100 nM Ac-DEVD-CHO (K_(i) caspase-1=17 nM; K_(i)caspase-3=0.2 nM; K_(i) caspase-6=31 nM; K_(i) caspase-7=1 nM; K_(i)caspase-8=0.92 nM; K_(i) caspase-9=60 nM; K_(i) caspase-10=12 nM) (FIG.4, lane 3), but were unaffected by 100 nM Ac-YVAD-CHO (K_(i)caspase-1=0.6 nM; K_(i) caspase-3 and caspase-7>10 μM; K_(i)caspase-10=408 nM) (FIG. 4, lane 4).

In the presence of 12.5 nM granzyme B, all three substrates wereefficiently cleaved (FIG. 4, lane 5). The predominant PARP fragmentinduced by granzyme B co-migrated with the fragment induced by caspase-3(FIG. 4, lane 5). There was also a minor (<5%) fragment of 62 kDa whichcorresponded with the fragment induced by granzyme B on purifiedsubstrate. In contrast, only a minor proportion of the NuMA fragmentsinduced by granzyme B co-migrated with the fragment generated bycaspase-3 (185 kDa). The major, novel fragment of 175 kDa (FIG. 4, lane5) corresponded with the fragment induced by granzyme B on purified invitro-translated substrate. Granzyme B-mediated cleavage of DNA-PK_(CS)generated a 250 kDa N-terminal fragment co-migrating with that generatedby caspase-3 (FIG. 4, lanes 2 & 5), as well as a unique 100 kDaC-terminal fragment which corresponded with that induced by granzyme Bon purified substrate (FIG.2, lane 2).

When Ac-DEVD-CHO or 2 mM iodoacetamide were added to the extracts 15minutes prior to the addition of granzyme B, cleavage of PARP was almostentirely abolished (FIG. 4, lane 6). In contrast, the cleavage of NuMAand DNA-PK_(CS) was only partially inhibited (FIG. 4, lane 6) (20-40%inhibition in 5 separate experiments). Generation of caspase-3-specificfragments of both NuMA (185 kDa) and DNA-PK_(CS) (150 kDa C-terminalfragment was abolished under these circumstances, while formation of the250 kDa N-terminal fragment of DNA-PK_(CS) was inhibited by 20-40% (FIG.4, lane 6).

Generation of granzyme B-specific fragments (175 kDa NuMA fragment and100 kDa C-terminal DNA-PK_(CS) fragment) was not affected by theinhibitors of caspase (FIG. 4, lane 6). The failure of Ac-DEVD-CHO tomarkedly inhibit generation of the 250 kDa DNA-PK_(CS) fragmentindicates that the majority of DNA-PK_(CS) cleavage detected afteraddition of granzyme B to lysates results from direct cleavage by thisprotease (rather than indirectly through activation of caspase-3). Takentogether, the results demonstrate that granzyme B competes withcaspase-3 for cleavage of endogenous substrates in cell lysates. Theoutcome of this competition can be accurately predicted by comparingk_(cat)/K_(m) values: for those substrates where k_(cat)/K_(m) forcleavage by caspase-3 is greater than that of granzyme B (e.g. PARP),caspase-3 fragments are detected almost exclusively. For thosesubstrates where the k_(cat)/K_(m) values are similar for the proteases,addition of granzyme B results in the formation of novel, granzymeB-specific fragments (e.g. DNA-PK_(CS) and NuMA).

EXAMPLE 6

Granzyme B Cleavage Sites in DNA-PK_(CS)

To address whether the 250 kDa DNA-PK_(CS) fragment generated bycaspase-3 and granzyme B result from cleavage at the same and/ordifferent, but closely-spaced sites, not distinguishable by SDS-PAGE, wegenerated a fragment of DNA-PK_(CS) (Met²⁵⁶⁶-Leu2928 SEQ ID NO: 34)encompassing both the known caspase-3 cleavage site at DEVD²⁷¹²-N²⁷¹³SEQ ID NO: 54 (Casciola-Rosen et al., 1996; Song et al., 1996b), as wellas the potential granzyme B cleavage site at VGPD²⁶⁹⁸-F²⁶⁹⁹ SEQ ID NO:34. The relevant P₁ aspartic acids (D²⁶⁹⁸ and D²⁷¹²) in this fragmentwere mutated to alanines (D²⁶⁹⁸A; D²⁷¹²A), and susceptibility of wildtype and mutated forms to cleavage by caspase-3 or granzyme B wasassessed (FIG. 5). Cleavage of the wild type protein by caspase-3resulted in two fragments of 26 kDa and 20 kDa (FIG. 5, lanes 1,2); thiscleavage was entirely abolished by the D²⁷¹²A mutation (FIG. 5, lanes7,8), confirming that caspase-3 cleaves at DEVD²⁷¹²-N²⁷¹³. In additionto 20 kDa and 26 kDa fragments identical to those generated bycaspase-3, granzyme B cleavage also resulted in fragments of 28 kDa and18 kDa (FIG. 5, lane 3); these unique fragments were enhanced by theD²⁷¹²A mutation (FIG. 5, lane 9), but were abolished by the D²⁶⁹⁸Amutation (FIG. 5, lane 6), placing a granzyme B cleavage site atVGPD²⁶⁹⁸-F²⁶⁹⁹SEQ ID NO: 34. These data demonstrate that granzyme B cancleave at VGPD²⁶⁹⁸-F²⁶⁹⁹ SEQ ID NO: 39, a site predicted by previousstudies using a combinatorial tetrapeptide substrate library (Thornberryet al., 1997). Furthermore, granzyme B also cleaves at the caspase-3cleavage site, DEVD²⁷¹²-N²⁷¹³ SEQ ID NO: 34. The C-terminal granzymeB-unique cleavage site is believed to be VDQD³²¹⁰-G³²¹¹ SEQ ID NO: 34.This cleavage yields the approximately 100 kDa fragment from amino acids3212 to 4096.

EXAMPLE 7 Granzyme B-Specific Fragments of DNA-PK_(CS) and NuMA areGenerated During Cytotoxic Lymphocyte Granule-Induced Target Cell Death.

To determine whether the granzyme B-specific fragments demonstrated incell lysates also occur when target cell death is induced by thecontents of cytolytic lymphocyte granules. The consequences ofgranule-induced target cell death on substrate cleavage were examined.Granules containing perforin and granzymes were purified from the humanNK cell line, YT. The granule contents were harvested and used to inducetarget cell cytotoxicity as described in Example 1. Several differenttarget cells (Jurkat T cells, K562 erythroleukemia cells, humanmyoblasts or HeLa cells) were incubated with granule contents (˜1.5×10⁷YT cell equivalents/ml) in the presence of Ca²⁺. Rapid target cell lysiswas induced (achieving ˜20%-40% specific ⁵¹Cr release in 60 min). Targetcell lysis by granule contents did not occur in the absence of Ca²⁺(Podack and Konigsberg, 1984; Young et al., 1986). DNA-PK_(CS), NuMA andPARP were all cleaved rapidly after addition of granules, in aCa²⁺-dependent manner (FIG. 6, lanes 2 & 3). As observed in the lysatesystem described above, cleavage of PARP resulted almost completely inthe generation of the 89 kDa caspase-3-specific fragment (FIG. 6, lane3). Only small amounts of the 62 kDa granzyme B-specific fragment weregenerated. Generation of the 89 kDa fragment was completely inhibited byAc-DEVD-CHO (FIG. 6, lane 4). In contrast, granule contents induced theformation of granzyme B-specific cleavage fragments of DNA-PK_(CS) andNuMA (FIG. 6, lane 3). The production of these fragments was notinhibited by Ac-DEVD-CHO (FIG. 6, lane 4).

Note that in the case of DNA-PK_(CS), addition of granule contents alsoresulted in the formation of small amounts of 150 kDa and 120 kDafragments, which were only well visualized after longer exposures of theX-ray film. Generation of these fragments was also entirely inhibited byAc-DEVD-CHO.

EXAMPLE 8

Granzyme B-Specific Fragments of DNA-PK_(CS) and NuMA are Generated inFas-Negative Target Cells Attacked by Lymphokine-Activated Killer (LAK)Cells.

To determine whether granzyme B-specific fragments were generated duringlymphocyte-induced cytotoxicity, we used the Fas-negative cell line K562as targets for LAK cells (McGahon et al. 1995; Topalian et al., 1989).To permit biochemical analysis of cleaved proteins in target cells,effector:target cell ratios of 5:1 were used. The signature 89 kDacaspase-3 fragment of PARP was generated during LAK-induced target celldeath (FIG. 7, lane 3). PARP cleavage was entirely abolished by 100 μMAc-DEVD-CHO (FIG. 7, lane 4). This was consistent with results observedwhen target cell death was initiated with YT cell granule contents (FIG.6, lane 4). Using anti-C-terminal antibodies, generation of both the 100kDa granzyme B-specific fragment of DNA-PK_(CS) and the 150-kDacaspase-3-specific fragment were detected during LAK-induced target celldeath (FIG. 7, lane 3). In addition, a 120 kDa fragment was observed inthese cells, consistent with the caspase-3-mediated cleavage ofDNA-PK_(CS) at DWVD-G previously observed both in vitro and in intactcells (Casciola-Rosen et al., 1996; Song et al., 1996b). While 100 μMAc-DEVD-CHO inhibited the generation of caspase-3-specific fragments ofDNA-PK_(CS) by >90%, the 100 kDa granzyme B-specific fragment wasinsensitive to this inhibitor (FIG. 7, lane 4). This data is consistentwith that obtained after initiating target cell death with YT cellgranule contents (FIG. 6, lane 4 and data not shown). Similarobservations were made for the granzyme B-specific NuMA fragment.Together, these data confirm that the novel fragments of DNA-PK_(CS) andNuMA defined in these studies are indeed generated when intactlymphocytes induce target cell cytotoxicity using the granule pathway.

EXAMPLE 9

Cytotoxic Lymphocyte Granules Induce Nuclear Morphologic Changes:Effects of Caspase Inhibitors.

Several of the downstream substrates for the caspases are directly andefficiently cleaved by granzyme B during lymphocyte granule-inducedcytotoxicity even in the presence of caspase inhibitors. It wasdetermined whether these granules also induced morphologic changes inthe target cell. HeLa cells were pre-incubated with YT cell granulecontents in the presence or absence of 100 μM Ac-DEVD-CHO, prior toaddition of Ca²⁺, and further incubation. YT cell granule contentsinduced the rapid onset (<60 min) of prominent surface blebbing (FIG.8A), followed by nuclear condensation and fragmentation intomembrane-bound apoptotic bodies (FIG. 8B). As described for UVB-inducedapoptosis (Casciola-Rosen et al. 1994), the autoantigens targeted insystemic autoimmune diseases are rapidly redistributed in target cellsexposed to YT cell granule contents, such that they become clusteredaround the rim of the condensing apoptotic nucleus (FIG. 8A), and thenultimately around apoptotic bodies (FIG. 8B). Granule content-inducedsurface blebbing, nuclear fragmentation, formation of apoptotic bodies,and characteristic redistribution of nuclear autoantigens was preventedby Ac-DEVD-CHO (Compare FIG. 8B, 8C). However, a prominent diminution inthe size of the nucleus (which was accompanied by condensation ofchromatin) was induced by granule contents in Ac-DEVD-CHO-treated cells(FIG. 8C); these nuclear changes were not observed when cells wereincubated with Ac-DEVD-CHO alone.

EXAMPLE 10

Antibodies Against Autoantigens and Autoantigenic Fragments.

The present invention also relates to polyclonal and monoclonalantibodies raised in response to the autoantigenic fragments disclosedherein. An antibody is specific for an epitope of an autoantigenicfragment if one of skill in the art can use standard techniques todetermine conditions under which one can detect an autoantigenicfragment in a Western Blot of a sample from cells of a tissue. The blotcan be of a native or denaturing gel as appropriate for the epitope. Anantibody is highly specific for an autoantigenic fragment epitope if nononspecific background binding is visually detectable. An antibody canalso be considered highly specific for an autoantigenic fragment if thebinding of the antibody can not be competed by random peptides,polypeptides or proteins, but can be competed by the particularautoantigenic fragment, autoantigen, or peptides or polypeptides derivedtherefrom.

Autoantigenic fragments can be separated from other cellular proteins byuse of an immunoaffinity column made with monoclonal or polyclonalantibodies specific for the autoantigen. Additionally, polyclonal ormonoclonal antibodies can be raised against a synthetic peptide (usuallyfrom about 9 to about 25 amino acids in length) from a portion of anautoantigen or autoantigenic fragment. Monospecific antibodies arepurified from mammalian antisera containing antibodies reactive againstthe autoantigenic fragment or are prepared as monoclonal antibodiesusing the technique of Kohler and Milstein (1975, Nature 256: 495-497).Monospecific antibody as used herein is defined as a single antibodyspecies or multiple antibody species with homogenous bindingcharacteristics for the autoantigenic fragment. Homogenous binding asused herein refers to the ability of the antibody species to bind to aspecific antigen or epitope, such as those associated with theautoantigenic fragment, as described herein. Autoantigenicfragment-specific antibodies are raised by immunizing animals such asmice, rats, guinea pigs, rabbits, goats, horses and the like, with anappropriate concentration of autoantigenic fragment or a syntheticpeptide generated from a portion of the autoantigenic fragment with orwithout an immune adjuvant.

Preimmune serum is collected prior to the first immunization. Eachanimal receives between about 0.1 mg and about 1000 mg of autoantigenicfragment associated with an acceptable immune adjuvant. Such acceptableadjuvants include, but are not limited to, Freund's complete, Freund'sincomplete, alum-precipitate, water in oil emulsion containingCorynebacterium parvum and RNA. The initial immunization consists ofinjecting autoantigenic fragment or peptide fragment thereof, preferablyin Freund's complete adjuvant, at multiple sites either subcutaneously(SC), intraperitoneally (IP) or both. Each animal is bled at regularintervals, preferably weekly, to determine antibody titer. The animalsmay or may not receive booster injections following the initialimmunization. Those animals receiving booster injections are generallygiven an equal amount of autoantigenic fragment in Freund's incompleteadjuvant by the same route. Booster injections are given at about threeweek intervals until maximal titers are obtained. At about 7 days aftereach booster immunization or about weekly after a single immunization,the animals are bled, the serum collected, and aliquots are stored atabout −20° C.

Monoclonal antibodies (mAb) reactive with the autoantigenic fragment areprepared by immunizing inbred mice, preferably Balb/c, with theautoantigenic fragment. The mice are immunized by the IP or SC routewith about 1 mg to about 100 mg, preferably about 10 mg, of theautoantigenic fragment in about 0.5 ml buffer or saline incorporated inan equal volume of an acceptable adjuvant, as discussed herein. Freund'scomplete adjuvant is preferred. The mice receive an initial immunizationon day 0 and are rested for about 3 to about 30 weeks. Immunized miceare given one or more booster immunizations of about 1 to about 100 mgof the autoantigenic fragment in a buffer solution such as phosphatebuffered saline by the intravenous (IV) route. Lymphocytes, fromantibody positive mice, preferably splenic lymphocytes, are obtained byremoving spleens from immunized mice by standard procedures known in theart. Hybridoma cells are produced by mixing the splenic lymphocytes withan appropriate fusion partner, preferably myeloma cells, underconditions which will allow the formation of stable hybridomas. Fusionpartners can include, but are not limited to: mouse myelomas P3/NS1/Ag4-1; MPC-11; S-194 and Sp 2/0, with Sp 2/0 being preferred. The antibodyproducing cells and myeloma cells are fused in polyethylene glycol,about 1000 mol. wt., at concentrations from about 30% to about 50%.Fused hybridoma cells are selected by growth in hypoxanthine, thymidineand aminopterin supplemented Dulbecco's Modified Eagles Medium (DMEM) byprocedures known in the art. Supernatant fluids are collected formgrowth positive wells on about days 14, 18, and 21 and are screened forantibody production by an immunoassay such as solid phaseimmunoradioassay (SPIRA) using the autoantigenic fragment as theantigen. The culture fluids are also tested in the Ouchterlonyprecipitation assay to determine the isotype of the mAb. Hybridoma cellsfrom antibody positive wells are cloned by a technique such as the softagar technique of MacPherson, 1973, Soft Agar Techniques, in TissueCulture Methods and Applications, Kruse and Paterson, Eds., AcademicPress.

Monoclonal antibodies are produced in vivo by injection of pristineprimed Balb/c mice, approximately 0.5 ml per mouse, with about 2×10⁵ toabout 6×10⁶ hybridoma cells about 4 days after priming. Ascites fluid iscollected at approximately 8-12 days after cell transfer and themonoclonal antibodies are purified by techniques known in the art.

In vitro production of anti-autoantigenic fragment mAb is carried out bygrowing the hybridoma in DMEM containing about 2% fetal calf serum toobtain sufficient quantities of the specific mAb. The mAb are purifiedby techniques known in the art.

Antibody titers of ascites or hybridoma culture fluids are determined byvarious serological or immunological assays which include, but are notlimited to, precipitation, passive agglutination, enzyme-linkedimmunosorbent antibody (ELISA) technique and radioimmunoassay (RIA)techniques. Similar assays are used to detect the presence of theautoantigenic fragment in body fluids or tissue and cell extracts.

It is readily apparent to those skilled in the art that the hereindescribed methods for producing monospecific antibodies can be utilizedto produce antibodies specific for autoantigenic fragment peptidefragments, or full-length autoantigen.

Antibody affinity columns are made, for example, by adding theantibodies to Affigel-10 (Biorad), a gel support which is pre-activatedwith N-hydroxysuccinimide esters such that the antibodies form covalentlinkages with the agarose gel bead support. The antibodies are thencoupled to the gel via amide bonds with the spacer arm. The remainingactivated esters are then quenched with IM ethanolamine HCl (pH 8). Thecolumn is washed with water followed by 0.23 M glycine HCl (pH 2.6) toremove any non-conjugated antibody or extraneous protein. The column isthen equilibrated in phosphate buffered saline (pH 7.3) and the cellculture supernatants or cell extracts containing the autoantigenicfragment are slowly passed through the column. The column is then washedwith phosphate buffered saline until the optical density (A₂₈₀) falls tobackground, then the protein is eluted with 0.23 M glycine-HCl (pH 2.6).The purified autoantigenic fragment is then dialyzed against phosphatebuffered saline.

Levels of an autoantigenic fragment in cells and tissues is quantifiedby a variety of techniques including, but not limited to, immunoaffinityand/or ligand affinity techniques. Autoantigenic fragment affinity beadsor autoantigenic fragment-specific antibodies are used to isolate³⁵S-methionine labeled or unlabelled autoantigenic fragment. Labeledautoantigenic fragment is analyzed by SDS-PAGE. Unlabelled autoantigenicfragment is detected by Western blotting, ELISA or RIA assays employingeither autoantigenic fragment specific antibodies and/orantiphosphotyrosine antibodies.

Preferred antibodies that bind to an autoantigenic fragment but do notbind to the intact autoantigen or other fragments thereof. Examples ofpreferred antibodies are those that recognize a cryptic epitope revealedin the autoantigenic fragment, or an antibody that recognizes a terminalepitope present only in the autoantigenic fragment.

EXAMPLE 11

Assay for the Detection of an Autoimmune Condition.

The autoantigenic fragments produced and identified following theteaching of the present invention can be used in a assay to detect thepresence of an autoimmune condition. The condition can be the generationof autoantigenic fragments before a disease state evolves, the presenceof an autoimmune disease or the lessening of a disease.

The assay is performed on a sample derived from a patient. Mostcommonly, the sample will be a tissue sample. The presence ofautoantigenic fragments can be detected in situ or can be partiallypurified before conducting the assay.

To perform an assay within this invention one prepares an autoantigenicfragment. For example, one can prepare an autoantigenic fragment of DNAPK_(CS) by cleaving the protein with granzyme B. The autoantigenicfragment is then used to prepare a monoclonal or polyclonal antibodyusing any of the methods widely known and used in the art.

The antibody can then be used to qualify or quantify the amount ofautoantigenic fragment present in the sample. This can be done bynumerous techniques known in the art including using antibody detectablylabeled with ¹²⁵I, gold, enzyme or other known labels. Alternatively, adetectable label can be carried on a second antibody specific for thefirst. The amount of autoantigenic fragment found is quantitatively orqualitatively compared to the amount of found on control cells. A changein the former relative to the latter is indicative of whether anautoimmune disease state is present, is progressing or is reduced.

In an alternative form of the assay one can treat cells as describedherein and then isolate the autoantigenic fragments present in treatedand control cells. The preparations can be made as crude cell extracts,membrane or intracellular fractions of the cells or after purificationsteps, e.g., chromatography, precipitation or affinity isolation steps.Crude, partially or highly purified preparations can be analyzed forautoantigenic fragment content, e.g., by using antibodies specific forthe autoantigenic fragment.

In another form of the assay, an autoantigenic fragment is used todetermine the presence or absence of an autoantibody in a patient as anindication of the presence or absence of an autoimmune condition. Theuse of particular types or autoantigenic fragments can also indicate thetype of autoimmune condition. The autoantibody to be assayed for can bepresent in the serum or a tissue sample of the patient. An autoantibodycan be detected in situ or after some purification of immunoglobins fromthe patient. In one format of the assay, the autoantigenic fragment canbe fixed to a support, an autoantibody present in a sample is thencontacted with the fragment to permit binding of the autoantibody to theautoantigenic fragment. After appropriate washing, the presence of boundautoantibody can be detected by methods available in the art, includingthe use of a labeled second antibody against the antibodies from thepatient.

In any assay it can be advantageous to devise an internal control sothat the results of different runs of assays can be compared to eachother. A cellular protein that is unrelated to the autoantigenicfragment and present in relatively constant amounts in the cells used inthe assay can serve as an internal control.

The assays described above are exemplary of all of the assays within thescope of the present invention. Those of skill in the art can use theautoantigenic fragments and antibodies of this invention in many assayformats known or developed in the art.

EXAMPLE 12

Tolerizing a Patient to the Presence of an Autoantigenic Fragment.

The present invention provides a method of tolerizing a patient to thefuture in vivo generation of compounds that are normally autoantigenic.This method can be prophylactic.

A patient diagnosed to be at risk of developing an autoimmune responseis identified. A sample of the tissue to which the autoimmune responseis possible is isolated from the patient. Autoantigenic fragments thatcan be generated from the tissue are then identified. The autoantigenicfragments are administered to the patient in pharmaceutically acceptablecarriers without an adjuvant to induce low-zone tolerance.

Tolerization typically involves purification of relevant autoantigenicfragments in a non-aggregated form. In particular embodiments,autoantigenic fragments of DNA Pk_(CS), NuMA or PARP are generated bythe action of granzyme B.

The autoantigenic fragments can also be present in a mixture. One suchmixture can be the product of the application of the contents ofgranules to a sample of tissue to which a potential autoimmune responseis diagnosed. In that case, the autoantigenic fragments are produced inthe mixture by the action of the granule contents, including granzyme B.

In any case, the autoantigenic fragments are administered at a low doseas chosen by a skilled physician or veterinarian to induce a low-zonetolerance in the patient. Once tolerization of the patient is achieved,if the normally autoantigenic fragments are produced in the tissue invivo, the immune system will not mount a response against them and theoccurrence of an autoimmune disease state can be avoided or the severityreduced.

EXAMPLE 13

Treatment for Malignant Cells.

The present invention also provides a method of generating an autoimmuneresponse against certain cells in a patient. For example, one can inducean autoimmune response against malignant cells in a patient that wouldbenefit from such a response.

For example, one can isolate a sample of malignant cells from a patientand contact the cells with the contents of granules or granzyme B. Theaction of the granule contents or granzyme B on the cells can produceautoantigenic fragments from autoantigens present in the cells. Theresulting mixture can then be administered to the patient. In this case,it is preferred that an adjuvant be administered with the autoantigenicfragments.

Those components of the mixture that are not altered to produce a newantigen will be recognized by the immune system as self molecules.However, those components that are altered to produce new autoantigenicfragments will be seen by the immune system as non-self and an immuneresponse will be generated against them.

Because the autoantigenic fragments produced in this way are the same asthose produced in vivo by the action of CTLs, NK and LAK cells on themalignant cells, the immune system is stimulated to generate a responseagainst the malignant cells. Therefore, the present invention provides amethod to heighten or stimulate the natural immune system processes toact against particular types of cells such as malignant cells. Themethod is particularly advantageous because the in vivo production ofthe autoantigenic fragments from, e.g., malignant cells, can occur atrates too low to stimulate the immune system, or at rates that can leadto a tolerization of the immune system.

EXAMPLE 14

Pharmaceutical Compositions.

Pharmaceutically useful compositions comprising autoantigenic fragmentsof the present invention can be formulated according to known methodssuch as by the admixture of a pharmaceutically acceptable carrier.Examples of such carriers and methods of formulation can be found inRemington's Pharmaceutical Sciences. To form a pharmaceuticallyacceptable composition suitable for effective administration, suchcompositions will contain an effective amount of the inhibitor.

Therapeutic, prophylactic or diagnostic compositions of the inventionare administered to an individual in amounts sufficient to treat ordiagnose disorders. The effective amount can vary according to a varietyof factors such as the individual's condition, weight, sex and age.Other factors include the mode of administration. The appropriate amountcan be determined by a skilled physician The pharmaceutical compositionscan be provided to the individual by a variety of routes such assubcutaneous, topical, oral and intramuscular.

The term “chemical derivative” describes a molecule that containsadditional chemical moieties which are not normally a part of the basemolecule. Such moieties can improve the solubility, half-life,absorption, etc. of the base molecule. Alternatively the moieties canattenuate undesirable side effects of the base molecule or decrease thetoxicity of the base molecule. Examples of such moieties are describedin a variety of texts, such as Remington's Pharmaceutical Sciences.

Compositions including autoantigenic fragments identified according tothe methods disclosed herein can be used alone at appropriate dosages.Alternatively, co-administration or sequential administration of otheragents can be desirable.

The present invention also provides a means to obtain suitable topical,oral, systemic and parenteral pharmaceutical formulations for use in themethods of treatment of the present invention. The compositionscontaining autoantigenic fragments identified according to thisinvention as the active ingredient can be administered in a wide varietyof therapeutic dosage forms in conventional vehicles for administration.For example, the compositions can be administered in such oral dosageforms as tablets, capsules (each including timed release and sustainedrelease formulations), pills, powders, granules, elixirs, tinctures,solutions, suspensions, syrups and emulsions, or by injection, asappropriate. Likewise, they can also be administered in intravenous(both bolus and infusion), intraperitoneal, subcutaneous, topical withor without occlusion, or intramuscular form, all using forms well knownto those of ordinary skill in the pharmaceutical arts.

Advantageously, autoantigenic fragments of the present invention can beadministered in a single daily dose, or the total daily dosage can beadministered in divided doses of two, three or four times daily.

Furthermore, compounds for the present invention can be administered inintranasal form via topical use of suitable intranasal vehicles, or viatransdermal routes, using those forms of transdermal skin patches wellknown to those of ordinary skill in that art. To be administered in theform of a transdermal delivery system, the dosage administration will,of course, be continuous rather than intermittent throughout the dosageregimen.

For combination treatment with more than one active agent, where theactive agents are in separate dosage formulations, the active agents canbe administered concurrently, or they each can be administered atseparately staggered times.

The dosage regimen utilizing compositions of the present invention isselected in accordance with a variety of factors including type,species, age, weight, sex and medical condition of the patient; whetherthe treatment is prophylactic or therapeutic; the severity of thecondition to be treated; the route of administration; the renal, hepaticand cardiovascular function of the patient; and the particular compoundthereof employed. A physician or veterinarian of ordinary skill canreadily determine and prescribe the effective amount of the drugrequired to prevent, counter or arrest the progress of the condition.Optimal precision in achieving concentrations of compounds of thisinvention, including purified autoantigenic fragments, within the rangethat yields efficacy without toxicity requires a regimen based on thekinetics of the compound's availability to target sites. This involves aconsideration of the distribution, equilibrium, and elimination of acompound.

EXAMPLE 15

Autoantigens Cleaved by Granzyme B

Granzyme B efficiently cleaves three caspase-3 substrates generatingunique fragments not generated during any other form of cell death. Todetermine whether the generation of unique autoantigen fragments bygranzyme B was a universal feature of autoantigens, a wide range ofautoantigens were tested for cleavage by granzyme B in vitro and invivo. It was determined that despite their diverse structure,distribution and function, >70% of the autoantigens described insystemic autoimmune diseases are efficiently cleaved by granzyme B andunique fragments are produced. In contrast, granzyme B does not generateunique fragments in all the non-autoantigen molecules tested. A panel ofautoantigens discovered to be susceptible to cleavage by granzyme B, arelisted in Table III along with the sites of cleavage.

The granzyme B cleavage sites in autoantigens were defined. In allcases, the tetrapeptide sequence immediately adjacent to the cleavagesite was highly conserved. The susceptibility to granzyme B cleavage istherefore a specific, unifying feature of these otherwise unrelatedmolecules. Furthermore, the ability of granzyme B to generate uniquefragments of these antigens indicates that granzyme B plays amechanistic role in selectively producing the fragments of thesemolecules against which autoimmune responses are initiated. Theseresults highlight a potential role for the cytotoxic lymphocytegranule-induced death pathway in initiation and propagation ofautoimmunity.

To determine whether susceptibility to direct cleavage by granzyme B wasan isolated feature of PARP, NuMA and DNA-PK_(CS) shown above, orwhether it was a more general feature of autoantigens, a variety ofwell-defined autoantigens selected from across the spectrum of systemicautoimmune diseases were tested for susceptibility to cleavage bypurified granzyme B. The efficiency of cleavage of the substrates bypurified granzyme B was also noted (Table III). Initially, a series ofautoantibodies of well-defined specificity were used to immunoblotlysates of HeLa cells that had been incubated in vitro with or withoutgranzyme B. Lysates were pre-treated with iodoacetamide (IAA) to preventinterference by endogenous caspase activity.

Interestingly, several autoantigens that have previously been shown tobe cleaved by caspases during apoptosis were also efficiently cleaved bygranzyme B. These substrates included U1-70 kDa, topoisomerase-1,SRP-72, PARP and NOR-90. In each case, unique fragments were generated.Mi-2, PMS2 and Ki-67 were also identified as additional autoantigensthat are cleaved into distinct, different sets of fragments both bycaspases and by granzyme B in these lysates.

Efficient cleavage and generation of novel fragments was also confirmedusing purified granzyme B to cleave in vitro translated substrates inthe case of U1-70 kDa, PARP, topoisomerase-1, PMS2, and Mi-2. Thecleavage efficiency (k_(cat)/K_(m)) of those substrates by granzyme Bwas determined using defined amounts of purified protease to cleaveeither endogenous substrates in cell lysates or radiolabeled substratesexpressed by in vitro transcription/translation, or both. Previousstudies have demonstrated that equivalent results are obtained usingeither form of substrate (Andrade et al., 1998). k_(cat)/K_(m) valuesvaried between 1.39×104 M⁻¹.s⁻¹ (PMS2) and 1.6×10⁶ M⁻¹.s⁻¹(topoisomerase-1) (see Table III).

Previous studies have identified several autoantigens that are notsusceptible to cleavage by caspases during apoptosis (Casciola-Rosen etal., 1995 & 1996). Many of these autoantigens were efficiently cleavedby granzyme B. These molecules included fibrillarin, PMS 1, CENP-B,Ku-70, La and RNA polymerase II large subunit. The efficiency ofcleavage of these substrates by granzyme B varied between 5.9×10³ M⁻¹.s⁻¹ (CENP-B) and 8×10⁴ M⁻¹.s⁻¹ (La) (See Table III). Interestingly,several ribonucleoprotein autoantigens were not susceptible to cleavageby either granzyme B or caspases. These included Ro52 kDa and 60 kDa,ribosomal protein P, histones and Sm proteins.

Susceptibility to cleavage by granzyme B was a highly specific featureof autoantigens. None of 18 different human non-autoantigens tested wascleaved by granzyme B. The precursors of caspases 3 and 7 are not knownto be autoantigens, but are efficiently cleaved by granzyme B.Interestingly, these substrates are cleaved at the same sites bygranzyme B and caspase-8, generating identical fragments (see below).

Since some well-defined autoantibodies do not recognize their antigensby immunoblotting, the susceptibility of radiolabeled endogenoussubstrates to cleavage by granzyme B in vitro was assessed. To performthese studies, HeLa cells were radiolabeled with [³⁵S]methionine/cysteine, and proteins were immunoprecipitated using humanautoantibodies. Protein A-agarose beads containing washed precipitatedproteins were resuspended in buffer supporting the activity of granzymeB, and incubated in the absence or presence of added purified granzymeB. Reaction products were visualized using SDS-PAGE and fluorography. Toconfirm the validity of this approach, several different autoantigensknown to be cleaved by granzyme B, as well as several autoantigens andnon-autoantigens that are not cleaved by granzyme B, were tested. Usingautoantibodies that both immunoblot and immunprecipitate, the cleavageprofiles obtained using granzyme B to cleave molecules in lysates(followed by detection with immunoblotting), and afterimmunoprecipitation (from [³⁵S]methionine-labeled HeLa lysate) werecompared. Identical results were obtained using these 2 methods forcleaved autoantigens (topoisomerase-1, Mi-2, RNA polymerase II largesubunit, Ku-70, PARP, and NOR-90), uncleaved autoantigens (Ku-80, Ro60k), and control substrates (□-tubulin, vinculin). Using thisimmunoprecipitation approach, it was demonstrated that severaladditional autoantigens (PMScl, RNA polymerase I large subunit, histidyltRNA synthetase, isoleucyl tRNA synthetase and alanyl tRNA synthetase)were cleaved by granzyme B, generating unique fragments (Table III). Ofnote, all these additional autoantigens (with the exception of RNApolymerase I) are targeted in autoimmune myositis. However, two othertRNA synthetases (threonyl tRNA synthetase and glycyl tRNA synthetase)that are also autoantigens in autoimmune myositis, were not cleavedusing this approach.

Thus, in addition to the three autoantigens described above to becleaved by both caspase-3 and granzyme B, these results identify anadditional 7 autoantigens have been identified that are cleaved by bothproteases but at different sites. Furthermore, another 10 autoantigensare cleaved exclusively by granzyme B, and not by caspases. Therefore,20 autoantigens targeted across the spectrum of human systemicautoimmune diseases are efficiently cleaved by granzyme B, generatingunique fragments not observed during other forms of cell death. (TableIII).

To confirm that similar autoantigen fragments are generated in intactcells during granule-induced cell death, K562 cells were exposed to YTcell granule contents in the presence of Ca²⁺, and the biochemicalstatus of the autoantigens were analyzed by immunoblotting. In thosecases where autoantigens are substrates for both caspases and granzymeB, both fragments were generated (U1-70 kDa, PARP, Mi-2,topoisomerase-1, Ki-67). Autoantigens known to be cleaved only bygranzyme B were indeed cleaved in the K562/YT granule system and thegranzyme B-specific fragments of Ku-70, PMS-1 and RNA polymerase IIlarge subunit were generated.

It was shown that granzyme B-specific fragments of DNA-PK_(CS) aregenerated during cytotoxic lymphocyte granule-induced target cell death.A similar approach was used to determine whether other autoantigenscleaved by granzyme B in vitro are also cleaved during killing of intactFas-negative target cells by lymphokine-activated killer (LAK) cells.Granzyme B-specific fragments of Mi-2, U1-70 kDa, topoisomerase-1, PMS-1and SRP-72 as well as Ku-70, RNA polymerase II and Ki-67, are generatedduring this form of cell death and were identified by immunoblottingwith appropriate antibodies. In the cases of Mi-2, U1-70 kDa, SRP-72 andtopoisomerase-1, which are all susceptible to direct cleavage by bothcaspase-3 and granzyme B, the amounts of granzyme B-specific fragmentsof these antigens appear to be determined by the relative efficiency ofcleavage by the two proteases. Thus, granzyme B-specific fragments ofMi-2 and topoisomerase-1 (which are efficiently cleaved by granzyme B)were clearly observed. In contrast, very low levels of granzymeB-specific fragments of U1-70 kDa, SRP-72 or the less efficientlycleaved sites on toposiomerase-1 (72 and 75 kDa) were observed in theintact cell killing assay unless caspases were inhibited by addingAc-DEVD-CHO. The cleavage of PMS1, which is a substrate for granzyme Bbut not for caspases, was unaffected by Ac-DEVD-CHO.

EXAMPLE 16

Specificity of Granzyme B Clevage of Autoantigens

Granzyme B is a serine protease whose specificity has been defined usinga positional scanning combinatorial tetrapeptide library. The proteasehas a preference for I,V or L in P₄, E, G, S in P₃, and P, S, N, A, Q,H, T, V, E, D, in P₂, with a preference for D in P₁. The sizes of thefragments generated by granzyme B cleavage and the cleavage specificitywas used to identify likely cleavage sites. Since granzyme B prefers Aspin the P₁ position, and does not tolerate Ala at that site,site-directed mutagenesis was employed to make a series of Asp-Alasubstitutions in several of the granzyme B substrates. The effects ofeach mutation on the efficiency of cleavage by the protease wasassessed.

The granzyme B cleavage sites in PARP and DNA-PK_(CS) have been defined.Using the above approach, the granzyme B cleavage sites in fibrillarin,Mi-2, topoisomerase 1, PMS1, PMS2, and U1-70 kDa were also defined(Table III). Interestingly, 10 of 11 of these cleavage sites contain P(7), A (2), or S (1) in the P₂ position, which are preferred by granzymeB but are poorly tolerated by group III caspases. Furthermore, 4cleavage sites also contain G or S in P₃. These residues are also nottolerated by group III caspases. Using fragment sizes to predict likelygranzyme B cleavage sites in other autoantigens, likely cleavage siteswere also identified in these proteins. In every case, these cleavagesites contained residues in P₂ and/or P₃ which are preferred by granzymeB, but are not tolerated by group III caspases (Table III).

To confirm that these substrates were not cleaved by group III caspases,the substrates were incubated with 50 nM purified caspase-8. Cleavageassays were performed in HeLa cell lysates in which endogenous caspaseshad first been irreversibly inactivated by 1 mM iodoacetamide (4° C. for15 min), prior to addition of 5 mM DTT to facilitate exogenous caspase-8activity. No cleavage of topoisomerase-1, Mi-2, U1-70 kDa, PARP, Ku-70,RNA polymerase II large subunit, SRP-72, NuMA or Ki-67 occurred.Caspase-8 was indeed active in these lysates, as evidenced by robustcaspase-3 cleavage products of PARP and U1-70 kDa seen when the IAA‘poisoning’ step was omitted. Radiolabeled PMS1 and PMS2 (generated byIVTT) were also not cleaved by purified caspase-8.

It was demonstrated that 20 of 28 autoantigens tested were susceptibleto efficient cleavage by granzyme B, generating unique fragments.Although the cleaved molecules differ markedly in subcellular location,function, and extended primary sequence, they share 2 features: (i) allare autoantigens targeted by a high titer autoantibody response in humanautoimmune diseases, including SLE, Sjogren's syndrome, diffuse andlimited scleroderma, and autoimmune myositis; and (ii) molecules areunified by containing a granzyme B cleavage site not susceptible tocleavage by caspase-8 (see below). Interestingly, autoantibodies againstthe precursors of caspases 3 and 7 (which are cleaved by granzyme B andcaspase-8 cleave at the same sites generating identical fragments) havenot been found in >1000 autoimmune sera screened by immunoblotting.

The status of a molecule as an autoantigen and its unique susceptibilityto cleavage by granzyme B but not by caspase-8, are therefore highlyrelated (P<0.0001; Chi-square analysis). The positive predictive valueof susceptibility to unique cleavage by granzyme B and status as anautoantigen is 100% for these 48 substrates, while thenegative-predictive value is 73%, indicating that additional mechanismsplay a role in selection of some molecules as autoantigens. It isnoteworthy that most of the uncleaved molecules are nucleoproteincomplexes (e.g. components of nucleosomes and snRNPs).

The granzyme B cleavage sites in several molecules are highly conserved,even in drosophila and yeast. This striking conservation of sequence atgranzyme B cleavage sites in organisms in which cytotoxic lymphocyteshad not yet evolved implies that an important, as yet undefined functionis served by these regions. This new, extended family of granzyme Bsubstrates therefore provide a powerful tool with which to explore theevolution and biological functions of the aspartic acid-specificapoptotic proteases, and to probe the mechanisms of cytotoxic lymphocytegranule-mediated cell death.

Human systemic autoimmune diseases represent a highly complex diseasespectrum, with numerous variables affecting individual susceptibility,initiation, and tissue targets. By demonstrating that the autoantigenstargeted across the spectrum of these diseases are unified by theirsusceptibility to efficient cleavage by granzyme B, with the generationof unique fragments not generated during any other form of cell death,these studies focus attention on the role of the cytotoxic lymphocytegranule pathway in initiation of autoimmunity. Where substrates arecleaved by both caspases and granzyme B, generation of unique granzyme Bfragments is dependent on relative inhibition of the caspases. Withoutwishing to be bound by a particular theory, it is therefore proposedthat during pro-immune intracellular infections occurring in amicroenvironment in which caspase activity is under relative inhibition,production of unique granzyme B fragments is favored. In susceptibleindividuals in whom clearance of apoptotic material is impaired,suprathreshold amounts of these fragments accumulate and are effectivelycaptured and presented by dendritic cells. The resulting immune responseis directed against products of CTL granule-induced death, generating anautoamplifying injury characteristic of these self-sustaining diseases.

EXAMPLE 17

Screening for Candidate Agents for Treatment

The assays described herein can be adapted for screening for candidateagents for the prophylactic or therapeutic treatment of autoimmunedisease, cancer, or the symptoms of such diseases. In an exemplaryformat, a candidate agent is contacted with both an uncleavedautoantigen and the contents of a lymphocyte granule, a granule enzyme,or granzyme B. The granule enzyme or granzyme B can be prepared invarying degrees of purity. The autoantigen should be a substratecleavable by the granule contents or, if a purified or partiallypurified enzyme is used, a substrate for the particular enzyme. Oncecontacted, one can monitor the cleavage of the autoantigen intoautoantigenic fragments. If desired, one can run a control assay with nocandidate agent, or a known inhibitor of the enzyme, in parallel. Theproduction of autoantigenic fragments can be monitored by a variety ofmeans known in the art including antibody capture of the epitopesproduced through cleavage, the loss of epitopes that span the cleavagesite, separation of cleavage products through chromatography orelectrophoresis and other techniques known and used in the art ordeveloped subsequently in the art of detection. A screening assay can bequantitative or qualitative.

A candidate agent can be a chemical compound, organic or inorganic, or abiochemical compound including proteins, peptides, glyco-proteins orpeptides, polysaccharides or other macromolecules.

A candidate agent that decreases the rate or the amount of cleavage ofthe autoantigen to autoantigenic fragments is referred to as aninhibitor of the process. Candidate agents can be studied to determinetheir suitability for application in the treatment of animals and humansby methods and procedure recognized in the art of pharmaceuticalsciences. Those candidates which through testing are shown to haveappropriate efficacy and an acceptable safety profile are used in theprophylactic or therapeutic treatment of patients.

References

Andrade, F., Roy, S., Nicholson, D., Thornberry, N., Rosen, A., andCasciola-Rosen, L. 1998. Granzyme B Directly and Efficiently CleavesSeveral Downstream Caspase Substrates: Implications for CTL-InducedApoptosis Immunity 8:451-460.

Bach, J. F. and S. Koutouzov. (1997). New clues to systemic lupus.Lancet 350:11-11.

Beidler, D. R., Tewari, M., Friesen, P. D., Poirier, G., and Dixit, V.M. (1995). The baculovirus p35 protein inhibits Fas- and tumor necrosisfactor-induced apoptosis. J.Biol.Chem. 270, 16526-16528.

Bockenstedt, L. K., R. J. Gee, and M. J. Mamula. (1995). Self-peptidesin the initiation of lupus autoimmunity. J.Immunol. 154:3516-3524.

Bump, N. J., Hackett, M., Hugunin, M., Seshagiri, S., Brady, K., Chen,P., Ferenz, C., Franklin, S., Ghayur, T., Li, P., Licari, P., Mankovich,J., Shi, L. F., Greenberg, A. H., Miller, L. K., and Wong, W. W. (1995).Inhibition of ICE family proteases by baculovirus antiapoptotic proteinp35. Science 269, 1885-1888.

Burlingame, R. W., R. L. Rubin, R. S. Balderas, and A. N.Theofilopoulos. (1993). Genesis and evolution of antichromatinautoantibodies in murine lupus implicates T-dependent immunization withself-antigen. J.Clin.Invest. 91:1687-1696.

Casciola-Rosen, L. A., Anhalt, G. J., and Rosen, A. (1994a).Autoantigens targeted in systemic lupus erythematosus are clustered intwo populations of surface structures on apoptotic keratinocytes. J.Exp. Med. 179, 1317-1330.

Casciola-Rosen, L. A., Miller, D. K., Anhalt, G. J., and Rosen, A.(1994b). Specific cleavage of the 70-kDa protein component of the U1small nuclear ribonucleoprotein is a characteristic biochemical featureof apoptotic cell death. J.Biol.Chem. 269, 30757-30760.

Casciola-Rosen, L. A., Anhalt, G. J., and Rosen, A. (1995).DNA-dependent protein kinase is one of a subset of autoantigensspecifically cleaved early during apoptosis. J.Exp.Med. 182, 1625-1634.

Casciola-Rosen, L. A., Nicholson, D. W., Chong, T., Rowan, K. R.,Thornberry, N. A., Miller, D. K., and Rosen, A. (1996). Apopain/CPP32cleaves proteins that are essential for cellular repair: A fundamentalprinciple of apoptotic death. J.Exp.Med. 183, 1957-1964.

Casciola-Rosen, L. and A. Rosen. (1997). Ultraviolet light-inducedkeratinocyte apoptosis: A potential mechanism for the induction of skinlesions and autoantibody production in LE. Lupus 6:175-180.

Casiano, C. A., Martin, S. J., Green, D. R., and Tan, E. M. (1996).Selective cleavage of nuclear autoantigens during CD95(Fas/APO-1)-mediated T cell apoptosis. J.Exp.Med. 184, 765-770.

Chinnaiyan, A. M. and Dixit, V. M. (1996a). The cell-death machine.Curr.Biol. 6, 555-562.

Chinnaiyan, A. M., Hanna, W. L., Orth, K., Duan, H. J., Poirier, G. G.,Froelich, C. J., and Dixit, V. M. (1996b). Cytotoxic T-cell-derivedgranzyme B activates the apoptotic protease ICE-LAP3. Curr.Biol. 6,897-899.

Darmon, A. J., Ley, T. J., Nicholson, D. W., and Bleackley, R. C.(1996). Cleavage of CPP32 by granzyme B represents a critical role forgranzyme B in the induction of target cell DNA fragmentation.J.Biol.Chem. 271, 21709-21712.

Darmon, A. J., Nicholson, D. W., and Bleackley, R. C. (1995). Activationof the apoptotic protease CPP32 by cytotoxic T-cell-derived granzyme B.Nature 377, 446-448.

Deveraux, Q. L., Takahashi, R., Salvesen, G. S. and Reed, J. C. (1997).X-linked IAP is a direct inhibitor of cell-death proteases. Nature 38,300-304.

Diamond, B., J. B. Katz, E. Paul, C. Aranow, D. Lustgarten, and M. D.Scharff. (1992). The role of somatic mutation in the pathogenic anti-DNAresponse. Ann.Rev.Immunol. 10:731-757.

Duan, H., Orth, K., Chinnaiyan, A. M., Poirier, G. G., Froelich, C. J.,He, W.-W., and Dixit, V. M. (1996). ICE-LAP6, a novel member of theICE/Ced-3 gene family, is activated by the cytotoxic T cell proteasegranzyme B. J.Biol.Chem. 271, 16720-16724.

Fernandes-Alnemri, T., Armstrong, R. C., Krebs, J., Srinivasula, S. M.,Wang, L., Bullrich, F., Fritz, L. C., Trapani, J. A., Tomaselli, K. J.,Litwack, G., and Alnemri, E. S. (1996). In vitro activation of CPP32 andMch3 by Mch4, a novel human apoptotic cysteine protease containing twoFADD-like domains. Proc.Natl.Acad.Sci.USA 93, 7464-7469.

Froelich, C. J., Hanna, W. L., Poirier, G. G., Duriez, P. J., D'Amours,D., Salvesen, G. S., Alnemri, E. S., Earnshaw, W. C., and Shah, G. M.(1996a). Granzyme B perforin-mediated apoptosis of jurkat cells resultsin cleavage of poly(ADP-ribose) polymerase to the 89-kDa apoptoticfragment and less abundant 64-kDa fragment. Biochem.Biophys.Res.Commun.227, 658-665.

Froelich, C. J., Orth, K., Turbov, J., Seth, P., Gottlieb, R., Babior,B., Shah, G. M., Bleackley, R. C., Dixit, V. M., and Hanna, W. (1996b).New paradigm for lymphocyte granule-mediated cytotoxicity—Target cellsbind and internalize granzyme B, but an endosomolytic agent is necessaryfor cytosolic delivery and subsequent apoptosis. J.Biol.Chem. 271,29073-29079.

Ghayur, T., Hugunin, M., Talanian, R. V., Ratnofsky, S., Quinlan, C.,Emoto, Y., Pandy, P., Datta, R., Huang, Y., Kharbanda, S., Allen, H.,Kamen, R., Wong, W., and Kufe, D. (1996). Proteolytic activation ofprotein kinase C d by an ICE/CED-3-like protease induces features ofapoptosis. J.Exp.Med. 184, 2399-2404.

Greidinger, E. L., Miller, D. K., Yamin, T.-T., Casciola-Rosen, L., andRosen, A. (1996). Sequential activation of three distinct ICE-likeactivities in Fas-ligated Jurkat cells. FEBS Lett. 390, 299-303.

Gu, Y., Sarnecki, C., Fleming, M. A., Lippke, J. A., Bleakley, R. C.,and Su, M. S. S. (1996). Processing and Activation of CMH-1 by GranzymeB. J.Biol.Chem 271, 10816-10820.

Heusel, J. W., Wesselschmidt, R. L., Shresta, S., Russell, J. H., andLey, T. J. (1994). Cytotoxic lymphocytes require granzyme B for therapid induction of DNA fragmentation and apoptosis in allogeneic targetcells. Cell 76, 977-987.

Irmler, M., Thome, M., Hahne, M., Schneider, P., Hofmann, B., Steiner,V., Bodmer, J. L., Schröter, M., Burns, K., Mattmann, C., Rimoldi, D.,French, L. E., and Tschopp, J. (1997). Inhibition of death receptorsignals by cellular FLIP. Nature 388, 190-195.

Jacobson, M. D., Weil, M., and Raff, M. C. (1997). Programmed cell deathin animal development. Cell 88, 347-354.

Jans, D. A., Jans, P., Briggs, L. J., Sutton, V., and Trapani, J. A.(1996). Nuclear transport of granzyme B (fragmentin 2). Dependence onperforin in vivo and cytosolic factors in vitro. J.Biol.Chem. 271,30781-30789.

Krajewska, M., Wang, H. G., Krajewski, S., Zapata, J. M., Shabaik, A.,Gascoyne, R., and Reed, J. C. (1997). Immunohistochemical analysis of invivo patterns of expression of CPP32 (Caspase-3), a cell death protease.Cancer Res. 57, 1605-1613.

Krajewski, S., Gascoyne, R. D., Zapata, J. M., Krajewska, M., Kitada,S., Chhanabhai, M., Horsman, D., Berean, K., Piro, L. D., Fugier-Vivier,I., Liu, Y. J., Wang, H. G., and Reed, J. C. (1997). Immunolocalizationof the ICE/Ced-3-family protease, CPP32 (Caspase-3), in non-Hodgkin'slymphomas, chronic lymphocytic leukemias, and reactive lymph nodes.Blood 89, 3817-3825.

Lanzavecchia, A. (1995). How can cryptic epitopes trigger autoimmunity?J.Exp.Med. 181,1945-1948.

Liu, X., Zou, H., Slaughter, C., and Wang, X. (1997). DFF, aheterodimeric protein that functions downstream of caspase-3 to triggerDNA fragmentation during apoptosis. Cell 89, 175-184.

Mamula, M. J. (1993). The inability to process a self-peptide allowsautoreactive T cells to escape tolerance. J.Exp.Med. 177:567-571.

Martin, S. J., Amarante-Mendes, G. P., Shi, L. F., Chuang, T. H.,Casiano, C. A., O'Brien, G. A., Fitzgerald, P., Tan, E. M., Bokoch, G.M., Greenberg, A. H., and Green, D. R. (1996). The cytotoxic cellprotease granzyme B initiates apoptosis in a cell-free system byproteolytic processing and activation of the ICE/CED-3 family protease,CPP32, via a novel two-step mechanism. EMBO J. 15, 2407-2416.

Martin, S. J. and Green, D. R. (1995). Protease activation duringapoptosis: Death by a thousand cuts? Cell 82, 349-352.

McGahon, A. J., Nishioka, W. K., Martin, S. J., Mahboubi, A., Cotter, T.G. and Green, D. R. (1995). Regulation of the Fas apoptotic cell deathpathway by Abl. J. Biol. Chem. 270, 22625-22631.

Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., O'Rourke, K., Shevchenko,A., Ni, J., Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M.,Krammer, P. H., Peter, M. E., and Dixit, V. M. (1996). FLICE, a novelFADD-homologous ICE/CED-3-like protease, is recruited to the CD95(Fas/APO-1) death-inducing signaling complex. Cell 85, 817-827.

Nicholson, D. W., Ali, A., Thornberry, N. A., Vaillancourt, J. P., Ding,C. K., Gallant, M., Gareau, Y., Griffin, P. R., Labelle, M., Lazebnik,Y. A., Munday, N. A., Raju, S. M., Smulson, M. E., Yamin, T., Yu, V. L.,and Miller, D. K. (1995). Identification and inhibition of the ICE/CED-3protease necessary for mammalian apoptosis. Nature 376, 37-43.

Nicholson, D. W. and Thornberry, N. A. (1997). Caspases: Killerproteases. TIBS 22, 299-306.

Odake, S., Kam, C. M., Narasimhan, L., Poe, M., Blake, J. T.,Krahenbuhl, O., Tschopp, J., and Powers, J. C. (1991). Human and murinecytotoxic T lymphocyte serine proteases: Subsite mapping with peptidethioester substrates and inhibition of enzyme activity and cytolysis byisocoumarins. Biochemistry 30, 2217-2227.

Pinkoski, M. J., Winkler, U., Hudig, D., and Bleackley, R. C. (1996).Binding of granzyme B in the nucelus of target cells. Recognition of an80 kDa protein. J.Biol.Chem. 271, 10225-10229.

Podack, E. and Konigsberg, P. J. (1984). Cytolytic T cell granules.Isolation, structural, biochemical and functional characterization.J.Exp.Med. 160, 695-710.

Poe, M., Blake, J. T., Boulton, D. A., Gammon, M., Sigal, N. H., Wu, J.K., and Zweerink, H. J. (1991). Human cytotoxic lymphocyte granzyme B:Its purification from granules and the characterization of substrate andinhibitor specificity. J.Biol.Chem. 266, 98-103.

Quan, L. T., Tewari, M., O'Rourke, K., Dixit, V., Snipas, S. J.,Poirier, G. G., Ray, C., Pickup, D. J., and Salveson, G. S. (1996).Proteolytic activation of the cell death protease Yama/CPP32 by granzymeB. Proc.Natl.Acad.Sci.USA. 93, 1972-1976.

Radic, M. Z. and M. Weigert. (1994). Genetic and structural evidence forantigen selection of anti-DNA antibodies. Ann.Rev.Immunol. 12:487-520.

Ramage, P., Cheneval, D., Chvei, M., Graff, P., Hemmig, R., Heng, R.,Kocher, H. P., Mackenzie, A., Memmert, K., Revesz, L., and Wishart, W.(1995). Expression, refolding, and autocatalytic proteolytic processingof the interleukin-1b-converting enzyme precursor. J.Biol.Chem. 270,9378-9383.

Salemi, S., A. P. Caporossi, L. Boffa, M. G. Longobardi, and V. Barnaba.(1995). HIVgp120 activates autoreactive CD4-specific T cell responses byunveiling of hidden CD4 peptides during processing. J.Exp.Med.181:2253-2257.

Sarin, A., Williams, M. S., Alexander-Miller, M. A., Berzofsky, J. A.,Zacharchuk, C. M., and Henkart, P. A. (1997). Target cell lysis by CTLgranule exocytosis is independent of ICE/Ced-3 family proteases.Immunity 6:209-215.

Sercarz, E. E., P. V. Lehmann, A. Ametani, G. Benichou, A. Miller, andK. Moudgil. (1993). Dominance and crypticity of T cell antigenicdeterminants. Ann.Rev.Immunol. 11:729-766.

Sercarz, E. E. and S. K. Datta. (1994). Mechanisms of autoimmunization:perspective from the mid-90s. Curr.Opin.Immunol. 6:875-881.

Shi, L. F., Mai, S., Israel, S., Browne, K., Trapani, J. A., andGreenberg, A. H. (1997). Granzyme B (GraB) autonomously crosses the cellmembrane and perforin initiates apoptosis and GraB nuclear localization.J.Exp.Med. 185, 855-866.

Shresta, S., Maclvor, D. M., Heusel, J. W., Russell, J. H., and Ley, T.J. (1995). Natural killer and lymphokine-activated killer cells requiregranzyme B for the rapid induction of apoptosis in susceptible targetcells. Proc.Natl.Acad.Sci.USA 92, 5679-5683.

Simitsek, P. D., D. G. Campbell, A. Lanzavecchia, N. Fairweather, and C.Watts. (1995). Modulation of antigen processing by bound antibodies canboost or suppress class II major histocompatibility complex presentationof different T cell determinants. J.Exp.Med. 181:1957-1963.

Song, Q. Z., Burrows, S. R., Smith, G., Lees-Miller, S. P., Kumar, S.,Chan, D. W., Trapani, J. A., Alnemri, E., Litwack, G., Lu, H., Moss, D.J., Jackson, S., and Lavin, M. F. (1996a). Interleukin-lb-convertingenzyme-like protease cleaves DNA-dependent protein kinase in cytotoxic Tcell killing. J.Exp.Med. 184, 619-626.

Song, Q. Z., Lees-Miller, S. P., Kumar, S., Zhang, N., Chan, D. W.,Smith, G. C. M., Jackson, S. P., Alnemri, E. S., Litwack, G., Khanna, K.K., and Lavin, M. F. (1996b). DNA-dependent protein kinase catalyticsubunit: A target for an ICE-like protease in apoptosis. EMBO J. 15,3238-3246.

Srinivasula, S. M., Fernandes-Alnemri, T., Zangrilli, J., Robertson, N.,Armstrong, R. C., Wang, L. J., Trapani, J. A., Tomaselli, K. J.,Litwack, G., and Alnemri, E. S. (1996). The Ced-3/interleukin lbconverting enzyme-like homolog Mch6and the lamin-cleaving enzyme Mch2aare substrates for the apoptotic mediator CPP32. J.Biol.Chem. 271,27099-27106.

Srinivasula, S. M., Ahmad, M., Ottilie, S., Bullrich, F., Banks, S.,Wang, Y., Fernandes-Alnemri, T., Croce, C. M., Litwack, G., Tomaselli,K. J., Armstrong, R. C and Alnemri, E. S. (1997). FLAME-1, a novelFADD-like anti-apoptotic molecule that regulates Fas/TNFR1-inducedapoptosis. J.Biol.Chem. 272,18542-18545.

Talanian, R. V., Yang, X., Turbov, J., Seth, P., Ghayur, T., Casiano, C.A., Orth, K., and Froelich, C. J. (1997). Granule-mediated killing:Pathways for granzyme B-initiated apoptosis. J.Exp.Med. 186, 1323-1331.

Thome, M., Schneider, P., Hofmann, K., Fickenscher, H., Meinl, E.,Neipel, F., Mattmann, C., Burns, K., Bodmer, J. L., Schroiter, M.,Scaffidi, C., Krammer, P. H., Peter, M. E., and Tschopp, J. (1997).Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced bydeath receptors. Nature 386, 517-521.

Thompson, C. B. (1995). Apoptosis in the pathogenesis and treatment ofdisease. Science 267, 1456-1462.

Thornberry, N. A. and Molineaux, S. M. (1995). Interleukin-1 βconverting enzyme: a novel cysteine protease required for IL-1 βproduction and implicated in programmed cell death. Protein Science 4,3-12.

Thornberry, N. A., Ranon, T. A., Pieterson, E. P., Rasper, D. M.,Timkey, T., Garcia-Calvo, M., Houtzager, V. M., Nordstrom, P. A., Roy,S., Vaillancourt, J. P., Chapman, K. T., and Nicholson, D. W. (1997). Acombinatorial approach defines specificities of members of the caspasefamily and granzyme B—Functional, relationships established for keymediators of apoptosis. J.Biol.Chem 272, 17907-17911.

Topalian, S. L., Solomon, D. and Rosenberg, S. A. (1989). Tumor-specificcytolysis by lymphocytes infiltrating human melanomas. J. Immunol. 142,3714-3725.

Trapani, J. A., Browne, K. A., Smyth, M. J., and Jans, D. A. (1996).Localization of granzyme-B in the nucleus—A putative role in themechanism of cytotoxic lymphocyte-mediated apoptosis. J.Biol.Chem. 271 ,4127-4133.

Tschopp, J. (1994). Granzyme B. Methods Enzymol. 244, 80-87.

Wang, S. Y., Miura, M., Jung, Y. K., Zhu, H., Gagliardini, V., Shi, L.F., Greenberg, A. H., and Yuan, J. Y. (1996). Identification andcharacterization of Ich-3, a member of the interleukin-1b convertingenzyme (ICE)/Ced-3 family and an upstream regulator of ICE. J.Biol.Chem.271, 20580-20587.

Watts, C. and A. Lanzavecchia. (1993). Suppressive effect of an antibodyon processing of T cell epitopes. J.Exp.Med. 178:1459-1463.

White, E. (1996). Life, death, and the pursuit of apoptosis. Genes Dev.10, 1-15.

Xue, D. and Horvitz, H. R. (1995). Inhibition of the Caenorhabditiselegans cell-death protease CED-3 by a CED-3 cleavage site inbaculovirus p35 protein. Nature 377, 248-251.

Yamin, T. T., Ayala, J. M., and Miller, D. K. (1996). Activation of thenative 45-kDa precursor form of interleukin-1β-converting enzyme.J.Biol.Chem. 271, 13273-13282.

Young, J. D.-E., Hengartner, H., Podack, E., and Cohn, Z. A. (1986).Purification and characterization of a cytolytic pore-forming proteinfrom granules of cloned lymphocytes with natural killer activity. Cell44, 849-859. TABLE I Comparison of k_(cat)/K_(m) (catalytic constant)values for the cleavage of different substrates by granzyme B andcaspase-3. The data obtained in time-course experiments weredensitometrically scanned and used to calculate the % cleavage of eachsubstrate. These values were fitted to the first order rate equation [%substrate cleaved = 100 * (1_(-e-((kcat*[E]/Km)*time)))] to calculatek_(cat)/K_(m). Measurements for each protease-substrate combination wereperformed at 3 different protease concentrations, enabling experimentalvariations in k_(cat)/K_(m) to be assessed. k_(cat)/K_(m) (M⁻¹s⁻¹)Cleavage by Cleavage by Substrate Method of Detection granzyme Bcaspase-3 DNA-PK_(cs) Immunoblotting 2.5 ± 0.8 × 10⁶ 7.5 ± 0.8 × 10⁶NuMA Fluorography 5.4 ± 1.5 × 10⁵ 5.0 ± 1.0 × 10⁵ Caspase-7¹Fluorography 1.8 ± 0.6 × 10⁵ — Caspase-7¹ Immunoblotting 1.9 ± 0.6 × 10⁵— Caspase-3 Fluorography 3.6 ± 1.0 × 10⁴ — Caspase-3 Immunoblotting 2.3± 0.4 × 10⁴ — PARP Fluorography 2.3 ± 1.8 × 10⁴ 5.0 ± 0.2 × 10⁶¹The source of caspase 3 and 7 precursors used in immunoblotting wasTHP⁻¹ cytosol. Blotting antibodies were specific for either caspase 3 or7, respectively.

TABLE II Different fragments are detected after in vitro cleavage ofautoantigens with granzyme B versus caspase-3. The data obtained in FIG.2, using purified DNA-PK_(cs), [³⁵S]methionine labeled PARP, endogenousDNA-PK_(cs) and NuMA, and purified proteases, were used for thetabulation below. Fragments induced after cleavage with Likely granzymeB Of SEQ Substrate Granzyme B Caspase-3 Cleavage sites ID No.DNA-PK_(cs2) 100 kDa 150 kDa VDQD³²¹⁰-G³²¹¹ 29 DNA-PK_(cs3) 250 kDa 250kDa VGPD²⁶⁹⁸-F²⁶⁹⁹; 24 DEVD²⁷¹²-N²⁷¹³ 25 NuMA 175 kDa 185 kDaVLGD⁴¹¹-V⁴¹² 30 PARP  62 kDa  89 kDa VGPD⁵³⁷-S⁵³⁸ 27 (Froelich et al.,1996a)₂DNA-PK_(cs) fragments were detected by immunoblotting with monoclonalantibody 25-4 or patient sera A.G. and G.A. (which all recognize theC-terminuis).₃DNA-PK_(cs) fragments were detected by monoclonal antibody 18-2 (whichrecognizes the N-terminus).

TABLE III AUTOANTIGENS ARE EFFICIENTLY CLEAVED BY GRANZYME SEQ IDk_(cat)/ Fragments NO Autoantigen Cleavage Site K_(m(M) ^(−1.s−1)) (kDa)24 DNA-PKcs VGPD²⁶⁹⁸-F 2.5 ± 0.8 × 10⁶ 160, 100 2 Topoisomerase I IEDA¹⁵-f 1.6 ± 0.6 × 10⁶ 97, 72 3 NuMA VATD¹⁷⁰⁵-A 5.4 ± 1.4 × 10⁵ 175 4Mi-2 V DPD¹³¹²-Y 8.5 ± 1.9 × 10⁴ 75, 72, 48 5 La LEED²²⁰-A 6.1 ± 1.7 ×10⁴ 21, 28 6 PMS1 LTPD³¹³-K 6.9 ± 0.9 × 10⁴ 48 37 ISA D⁴⁹⁶-E 7Fibrilliarin VGPD¹⁸⁴-G 3.3 ± 1.9 × 10⁴ 20, 17 8 PARP VDPD⁵³⁶-S 2.3 ± 1.8× 10⁴ 72, 62, 55 9 U1-70 kDa LGND⁴⁰⁹-S 1.3 ± 0.4 × 10⁴ 60 10 PMS2VEKD⁴⁹³-S 1.4 ± 0.6 × 10⁴ 60, 45, 35 11 Isoleucyl tRNA VTPD982-Q 7.8 ±1.8 × 10⁴ synthetase (O.J.) 12 Histidyl tRNA LGPD⁴⁸-E 2.3 ± 0.7 × 10⁴ 40synthetase (Jo-1) 13 Alanyl tRNA VAPD⁶³²-R 1.8 × 10⁴ 63, 40 synthetase(PL-12) 14 RNA polymerase I ICPD⁴⁴⁸-M 1.3 ± 0.5 × 10⁴ 140 15 KI-67VCTD¹⁴⁸¹-K 8.1 ± 2.6 × 10³ 168, 148 16 PmScl VEQD²⁵²-M 7.5 ± 1.4 × 10³85, 74, 60 17 CENP B VDSD⁴⁵⁷-E 5.9 ± 0.2 × 10³ 58, 40 18 RNA polymeraseII I TPD³⁷⁰-P ND 200 19 SRP 72 VTPD⁵⁷³-P ND 62 20 Ku 70 I SSD⁷⁹-R ND 6521 Tyrosinase ICTD²⁴⁹ 22 E4 VDVD¹²³ 23 Golgin 160 SEVD³¹¹ 36 Golgin 160VGPD⁹² 2 Golgin 160 IEAD⁶⁴⁸ Myosin

1. A composition comprising at least one purified and isolatedautoantigenic fragment wherein said fragment is produced from anautoantigen of a pre-apoptotic cell by the action of at least onelymphocyte granule enzyme, wherein said fragment has at least oneterminus derived from the cleavage site of said enzyme.
 2. Thecomposition of claim 1 wherein the granule enzyme is Granzyme B.
 3. Thecomposition of claim 2 wherein the autoantigenic fragment is derivedfrom an autoantigen that is a substrate for a caspase and the fragmentis produced by the Granzyme B catalyzed cleavage of said protein at asite that is not cleaved by caspase.
 4. The composition of claim 2comprising an autoantigenic fragment produced by the Granzyme Bcatalyzed cleavage of an autoantigen selected from the group consistingof DNA PK_(CS), PARP and NuMA.
 5. The composition of claim 4 comprisingat least one autoantigenic fragment selected from the group consistingof DNA-PK_(CS) from amino acid 2699 to 4096; DNA-PK_(CS) from amino acid3211to 4096; PARP from amino acid 1 to 538; PARP from amino acid 538 to1004; NuMA from amino acid 412 to 2111 and NuMA from amino acid 1 to1799.
 6. A pharmaceutical composition comprising at least one purifiedand isolated autoantigenic fragment having at least one terminus derivedfrom a granule enzyme cleavage site, wherein said cleavage site is notcleaved by a caspase, and a pharmaceutically acceptable carrier.
 7. Thecomposition of claim 6 wherein the granule enzyme is granzyme B.
 8. Thecomposition of claim 6 wherein the autoantigenic fragment is selectedfrom the group of fragments consisting of DNA-PK_(CS) from amino acids2699 to 4096; DNA-PK_(CS) from amino acids 3211 to 4096; PARP from aminoacid 1 to 538; PARP from amino acids 538 to 1004; NuMA from amino acids412 to 2111 and NuMA from amino acids 1 to
 1799. 9. The pharmaceuticalcomposition of claim 6 comprising at least one autoantigenic fragmentderived from a malignant cell.
 10. A method of treating a patient inneed of treatment for an autoimmune disease comprising administering atleast one autoantigenic fragment of claim
 1. 11. The method of claim 10wherein the treatment is prophylactic.
 12. The method of claim 10wherein the autoantigenic fragment is selected from the group offragments consisting of DNA-PK_(CS) from amino acids 2699 to 4096;DNA-PK_(CS) from amino acids 3211 to 4096; PARP from amino acids 538 to1004; NuMA from amino acids 412 to 2111 and NuMA from amino acids 1 to1799.
 13. The method of claim 10 wherein the method is a method oftolerizing said patient to the presence of said fragment comprising thesteps of: (a) identifying a target tissue and isolating cells from thetissue, (b) providing at least one lymphocyte granule enzyme, (c)contacting the cells with said at least one lymphocyte granule enzyme toproduce at least one autoantigenic fragment, (d) administering said atleast one autoantigenic fragment to the patient.
 14. The method of claim13 wherein said at least one lymphocyte granule enzyme provided in step(b) is isolated from the contents of a lymphocyte granule.
 15. Themethod of claim 10 for the therapeutic treatment of a patient producingautoantigenic fragments and autoantibodies against the fragmentscomprising the steps of: (a) providing an isolated autoantigenicfragment associated with the autoimmune condition in the patient, (b)contacting the serum of the patient with the autoantigenic fragmentunder conditions that allow the binding of autoantibodies to saidautoantigenic fragment.
 16. The method of claim 15 wherein at least aportion of the autoantibodies are removed from the serum of the patient.17. The method of claim 15 wherein the autoantibodies are bound to theisolated autoantigenic fragment in vivo.
 18. A method of treating apatient in need of treatment for a malignancy comprising the steps of(a) providing at least one enzyme of a lymphocyte granule, (b) isolatingmalignant cells from the patient, (c) contacting the malignant cellswith the enzyme to produce a mixture containing autoantigenic fragments,and (d) administering the autoantigenic fragments to the patient.
 19. Anassay for the detection of an autoantigenic fragment in a patient as anindication of the presence or absence of an autoimmune condition in apatient comprising: (a) providing a sample from the patient, (b)contacting the sample with an antibody that specifically binds to acryptic epitope of an autoantigenic fragment, said fragment having atleast one terminus derived from a granule enzyme cleavage site, (c)detecting the presence or absence of the binding of the antibody to theautoantigenic fragment as an indication of the presence or absence of anautoimmune condition in a patient.
 20. The assay of claim 19 wherein thegranule enzyme is granzyme B.
 21. An assay for the detection of anantibody that binds an autoantigenic fragment as an indication of thepresence or absence of an autoimmune condition in a patient comprising:(a) providing a sample from the patient, (b) contacting the sample withan autoantigenic fragment having at least one terminus derived fromcleavage by a granule enzyme, (c) detecting the presence or absence ofthe binding of an antibody in the sample to the autoantigenic fragmentas an indication of the presence or absence of an autoimmune conditionin the patient.
 22. The assay of claim 21 wherein the granule enzyme isgranzyme B.
 23. A method of making an autoantigenic fragment from anautoantigen comprising the steps of (a) isolating cells containing atleast one autoantigen, and (b) contacting the cells with a lymphocytegranule enzyme to produce a mixture containing at least oneautoantigenic fragment.
 24. The method of claim 22 further comprisingthe step of (c) isolating said at least one autoantigenic fragment. 25.The method of claim 22 wherein step (a) further comprises purifying atleast one autoantigen from the cells and step (b) comprises contactingsaid purified autoantigens with granzyme B.
 26. The method of claim 25wherein in step (a) the at least one autoantigen is at least one ofDNA-PK_(CS), PARP and NuMA, and step (b) comprises contacting said atleast one autoantigen with granzyme B.
 27. The method of claim 22wherein said lymphocyte granule enzyme is isolated from the granules ofat least one lymphocyte selected from the group consisting of cytotoxicT lymphocytes (CTL), natural killer cells (NK), lymphokine activatedkiller cells (LAK) and cells of the YT cell line.
 28. A method ofidentifing candidate agents for preventing or treating autoimmunedisease symptoms comprising: a) contacting a test substance with atleast one granule enzyme and an autoantigen which is a substrate forsaid at least one granzyme enzyme; b) monitoring the cleavage of theautoantigen said enzyme into autoantigenic fragments; c) determiningwhether the candidate agent alters the production of the autoantigenicfragments; wherein a test substance which inhibits the cleavage isidentified as a candidate agent for treating autoimmune diseases. 29.The method of claim 28 wherein the granule enzyme is granzyme B.